University of Southern California Dissertations and Theses

Characterization of the interaction of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 12 (Nlrp12) with hematopoietic cell kinase (Hck)

(USC Thesis Other)

Characterization of the interaction of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 12 (Nlrp12) with hematopoietic cell kinase (Hck)

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

CHARACTERIZATION OF THE INTERACTION OF
NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN,
LEUCINE-RICH REPEAT AND PYRIN DOMAIN-
CONTAINING PROTEIN 12 (NLRP12) WITH
HEMATOPOIETIC CELL KINASE (HCK)

by

Yue Zhang

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

December 2019

Copyright 2019 Yue Zhang
ii

Dedication
This dissertation is dedicated in
memory of my father and a physician
Yuanbo Zhang (Jan. 16
th
, 1959-Mar. 18
th
, 2019)

iii

Table of Contents
Dedication .................................................................................................................................. ii
List of Figures .......................................................................................................................... vi
List of Tables .......................................................................................................................... viii
Abbreviations ............................................................................................................................ ix
Acknowledgements ................................................................................................................ xvii
Abstract ................................................................................................................................... xix
Chapter 1: Nlrp12: a multifunctional protein-effects on immune responses to gut microbiota . 1
Nlrp12 and regulation of NF-κB pathways .......................................................................... 3
Nlrp12 and autoimmune diseases ......................................................................................... 5
Nlrp12 and autoimmune models ........................................................................................ 10
Nlrp12 and colitis-induced colon cancer ............................................................................ 11
Nlrp12’s role in regulating gut microbiota ........................................................................ 12
Nlrp12 and bone physiology ............................................................................................... 14
Nlrp12 is correlated with prostate cancer .......................................................................... 14
Nlrp12 in other functional roles ......................................................................................... 15
Conclusion ........................................................................................................................... 16
Chapter 2: Hematopoietic cell kinase (Hck)’s structure and functions ................................... 17
Hck structure ...................................................................................................................... 17
Hck’s functions ................................................................................................................... 19
Hck’s function in leukemia ............................................................................................. 19
Hck and inhibitors related to leukemia .......................................................................... 21
Chapter 3: Material and Methods ............................................................................................ 25
Reagents and cells ............................................................................................................... 25
Cloning ................................................................................................................................ 26
PCR ..................................................................................................................................... 34
Yeast two-hybrid assay ....................................................................................................... 34
Yeast transformed with the plasmid PGBKT7-Nlrp12’s PYD + NBD ......................... 34
Library scale transformation of cDNA library into yeast that have been transformed
with the PGBKT7-Nlrp12 PYD + NBD plasmid ............................................................ 35
Small scale transformation ............................................................................................. 35
Docking analysis ................................................................................................................. 36
Cell transfection .................................................................................................................. 36
Nlrp12 stable cell lines ........................................................................................................ 37
iv

Knock-down cells ................................................................................................................ 39
Cell lysis and immunoprecipitation ................................................................................... 39
Immunoblotting .................................................................................................................. 40
Immunofluorescence ........................................................................................................... 41
Ingenuity Pathway Analysis (IPA) diagram ...................................................................... 42
Bioinformatic analysis ........................................................................................................ 42
Luciferase assay .................................................................................................................. 42
Real-time (RT)-PCR ........................................................................................................... 43
AlamarBlue® assay ............................................................................................................ 45
Cell counting using a hemocytometer ................................................................................ 45
Mice work and blood collection .......................................................................................... 45
Flowcytometry .................................................................................................................... 46
Statistical analysis ............................................................................................................... 48
Chapter 4: Nucleotide binding domain and leucine-rich repeat pyrin domain-containing
protein 12: characterization of its binding to hematopoietic cell kinase .................................. 49
Introduction ........................................................................................................................ 49
Results ................................................................................................................................. 52
Nlrp12 interacts with Hck in the yeast two-hybrid assay .............................................. 52
In directed screens using the yeast two-hybrid assay, Nlrp12’s PYD + NBD domain
binding to Hck C-terminal 40 amino acids appears to be selective for Nlrp12 and Hck
binding to Nlrp12 appears to be selective for Hck ......................................................... 59
In directed screens using the yeast two-hybrid assay, amino acids F503, Q507, L510,
and D511 of Hck are critical for the binding between Nlrp12’s PYD + NBD and Hck
C-terminal 40 amino acids .............................................................................................. 63
In directed screens using the yeast two-hybrid assay, the shortest fragment of Hck that
binds to Nlrp12’s PYD + NBD is its C-terminal 30 amino acids ................................... 69
Nlrp12 co-immunoprecipitates with Hck from mammalian cells, and Nlrp12 is co-
localized with Hck in mammalian cells .......................................................................... 72
Docking of the Hck 5 amino acids to the Nlrp12’s PYD + NBD domains ..................... 78
Survival rates of AML in some of acute myeloid leukemia patients are related to Hck
......................................................................................................................................... 84
In blood and marrow samples from patients with AML, Nlrp12 and Hck co-occur and
are co-expressed .............................................................................................................. 89
Other potential binding partners of Nlrp12 from the yeast two-hybrid screen ........... 93
Discussion ............................................................................................................................ 96
Chapter 5: Characterization of the functional consequences of Nlrp12’s interaction with Hck
............................................................................................................................................... 101
Hypothesis 1: Nlrp12 and Csk compete with each other for binding to Hck’s C-terminus
to regulate Hck activity..................................................................................................... 101
v

Introduction .................................................................................................................. 101
Results and Discussion .................................................................................................. 102
Hypothesis 2: The interaction of Nlrp12 with Hck causes or prevents Nlrp12 degradation
and/or Hck degradation. ................................................................................................... 108
Introduction .................................................................................................................. 108
Results and discussion .................................................................................................. 110
Hypothesis 3: Nlrp12 is a p-Tyr substrate of Hck. .......................................................... 120
Introduction .................................................................................................................. 120
Results and discussion .................................................................................................. 126
Hypothesis 4: Hck modulates Nlrp12-mediated inhibition of NF-κB activity. ............... 128
Introduction .................................................................................................................. 128
Result and discussion .................................................................................................... 130
Hypothesis 5: Nlrp12 inhibits or activates Hck activation. ............................................. 142
Introduction .................................................................................................................. 142
Result and discussion .................................................................................................... 143
Hypothesis 6: Nlrp12 and Hck impact proliferation rates in U937 and K562 human
leukemia cell lines ............................................................................................................. 152
Introduction .................................................................................................................. 152
Result and discussion .................................................................................................... 153
Conclusions for all the six hypotheses .............................................................................. 156
Chapter 6: Mice work on acute myeloid leukemia ................................................................. 157
Introduction ...................................................................................................................... 157
Results and discussions ..................................................................................................... 157
Chapter 7: Conclusions ......................................................................................................... 161
Chapter 8: Future Directions ................................................................................................ 162
1. Docking experiments..................................................................................................... 162
2. Hypothesis 1: Nlrp12 and Csk compete with each other for binding to Hck’s C-
terminus to regulate Hck activity ..................................................................................... 165
3. Hypothesis 2: The interaction of Nlrp12 with Hck causes or prevents Nlrp12
degradation and/or Hck degradation. .............................................................................. 165
4. Hypothesis 3: Nlrp12 is a p-Tyr substrate of Hck. ...................................................... 166
5. Hypothesis 4: Hck modulates Nlrp12-mediated inhibition of NF-κB activity. ........... 167
6. Hypothesis 5: Nlrp12 inhibits or activates Hck activation. ......................................... 168
7. Hypothesis 6: Nlrp12 and Hck impact proliferation rates in U937 and K562 human
leukemia cell lines ............................................................................................................. 169
Chapter 9: Bibliography ........................................................................................................ 170

vi

List of Figures

Figure 1: Canonical and non-canonical NF-κB pathways. ..................................................... 7
Figure 2: Nlrp12 and its polymorphisms. ................................................................................ 9
Figure 3: Two isoforms of Hck and schematic drawing of the structure of Hck.. ............... 23
Figure 4: Hck crystal structure (picture derived from pMOL software) (protein data bank
(PDB) accession number is 1AD5). ........................................................................................ 24
Figure 5: Yeast two-hybrid prey, bait, and results. ............................................................... 54
Figure 6: Specificity of binding of Nlrp12 with Hck.. ........................................................... 61
Figure 7: Four amino acids in Hck C terminals are critical for binding to Nlrp12’s PYD +
NBD domain. .......................................................................................................................... 66
Figure 8: The last 30 amino acids of Hck C terminal are critical for binding to Nlrp12’s
PYD + NBD domains. ............................................................................................................. 70
Figure 9: Nlrp12 co-immunoprecipitates with Hck and co-localizes with Hck by
immunofluorescence. .............................................................................................................. 74
Figure 10: Immunofluorescent image of epitope-tagged Nlrp12-3FLAG and Hck co-
transfected into 293T cells, and fluorescently immunolabeled with anti-Hck and anti-FLAG
(Nlrp12) antibodies, and counterstained with DAPI.. ........................................................... 76
Figure 11: Docking of Hck’s 5 amino acid fragment Gln507-Ser-Val-Leu-Asp511 to
Nlrp12’s PYD + NBD.............................................................................................................. 82
Figure 12: The red curves mean that Hck is highly expressed. The black lines mean that
Hck expression level is lower. ................................................................................................. 87
Figure 13: Nlrp12 is co-expressed with Hck in AML patient samples. ................................ 92
Figure 14: IPA graph.. ............................................................................................................ 94
Figure 15: Schematic drawing of the possible relationship between Nlrp12, Csk, and Hck
(Hypothesis 1).. ..................................................................................................................... 105
Figure 16: Cell lysis and immunoprecipitation of Hck in 293T cells overexpressing Nlrp12-
V5, Hck, and Csk.. ................................................................................................................ 106
Figure 17: The co-expression of Hck protein significantly decreased the steady-state protein
expression levels of Nlrp12.. ................................................................................................. 112
Figure 18: The stability of Nlrp3 vs. Nlrp12 in the presence of increasing amounts of Hck
expression plasmid.. .............................................................................................................. 114
Figure 19: Overexpression of members of the Nlrp family of proteins and Hck in 293T
cells.. ...................................................................................................................................... 116
Figure 20: Co-expression of Hck and Nlrp12 showed no change in Hck expression level,
although the transfected amount of expression plasmid for Nlrp12 was increased. .......... 118
Figure 21: The alignment of Nlrp12 from mammalian species showing that tyrosine
residues Tyr16, Tyr129, Tyr132, Tyr157, Tyr246, Tyr377, Tyr435, Tyr438, Tyr548,
Tyr608, Tyr671, Tyr702, and Tyr1057 (these amino acids are underlined and colored red)
are common in all of the species. .......................................................................................... 124
Figure 22: Epitope-tagged Nlrp12 and Hck were co-transfected in 293T cells.. ................ 127
Figure 23: Analysis of cytokine changes by RT-PCR in different stable RAW 264.7 cell
lines (i.e., cells transfected with empty vector (pEFIRES-P-puro); cells transfected with
Nlrp12-3FLAG (pEFIRES-P-puro-Nlrp12-3FLAG); cells transfected with Hck (pEFIRES-
vii

P-hygro-Hck); and cells co-transfected with Nlrp12-3FLAG and Hck (pEFIRES-P-puro-
Nlrp12-3FLAG and pEFIRES-P-hygro-Hck)). ................................................................... 132
Figure 24: Activation of the canonical NF-κB pathway by stimulation of pro-monocytic
U937 cells, as assayed by determining the ratio of phospho-IκB-α to IκB-α. ..................... 136
Figure 25: Duo-Glo luciferase assay to detect whether Hck affected Nlrp12-inhibited NF-
κB activation. ........................................................................................................................ 140
Figure 26: Immunoprecipitation of Hck from U937 cells showed that the phosphorylation
level of Hck in the immunoprecipitates was decreased when Nlrp12 is co-expressed and co-
immunoprecipitated.............................................................................................................. 145
Figure 27: Hck phosphorylation is decreased in Hck immunoprecipitates when Nlrp12 is
co-immunoprecipitated. ........................................................................................................ 147
Figure 28: Quantification of the pHck protein expression levels to Hck protein expression
levels from Figures 26 and 27.. ............................................................................................. 149
Figure 29: Immunoprecipitation of Hck from U937 cells treated with 1 µg/ml of Lps. ..... 150
Figure 30: Cell proliferation performed by AlamarBlue® assay and manual cell counting
to compare the proliferation rates of Nlrp12-expressing U937 cells. .................................. 155
Figure 31: Comparison of the neutrophil and monocyte counts between WT mice and
Nlrp12 -/- mice. ..................................................................................................................... 159
Figure 32: The gating strategies for neutrophils and monocytes.. ...................................... 160

viii

List of Tables

Table 1: Primers used in cloning (The underline shows the restriction enzymes) ............... 29
Table 2: Antibodies added into the flow cytometry buffer to detect blood leukocyte
composition ............................................................................................................................. 47
Table 3: Complete yeast two hybrid results .......................................................................... 55
Table 4: Yeast two-hybrid results (hit frequency >=3) ......................................................... 58
Table 5: Co-occurrence of mRNA expression level of Hck and all of the Nlrp family
proteins (from the provisional data, assessed on July 11 2019) ............................................ 90
Table 6: Substrates of Hck (continue (Poh et al., 2015) with modifications) ...................... 122

ix

Abbreviations

3-AT: 3-amino 1, 2, 4-triazole
ALA: Alanine
ALL: Acute lymphoblastic leukemias
AML: Acute myeloid leukemia
ANOVA: Analysis of variance
AOM: Azoxymethane
AP1: Activator protein 1 (a.k.a. c-Jun)
APL: Acute promyelocytic leukemia
ARG: Arginine
ASC: Apoptosis-associated speck-like protein containing a caspase activation and recruitment
domain
ASN: Asparagine
ASP (D): Aspartic acid
ATCC: American Type Culture Collection
ATP: Adenosine triphosphate
BAFF: B cell-activating factor
BCL/ABL: Breakpoint cluster region protein/Abelson murine leukemia
BD: Becton-Dickinson
C1 or C1 fragment: Self-named truncations of C-terminal 40 (or 42) amino acids fragment of Src
family non-receptor tyrosine kinases
C2-C7 and N1-N5: Self-named truncations Hck C1 fragment
x

CARD: Caspase activation and recruitment domain
CBL: Casitas B-lineage lymphoma
CCL11: C-C motif chemokine ligand 11
CD: Cluster of differentiation (CD45: a. k. a. protein tyrosine phosphatase receptor type C)
CDK6: Cyclin-dependent kinase 6
CFU: Colony-forming units
CHK: Checkpoint kinase
CLRs: C-type lectin receptors
CML: Chronic myeloid leukemia
CMV: Cytomegalovirus
COSMIC: Catalogue of Somatic Mutations in Cancer
CRC: Colorectal cancer
CsCl: Cesium Chloride
CSK: C-terminal Src kinase
CXCL: The chemokine (C-X-C motif) ligand (CXCL) (CXCL13: a. k. a. B cell attracting
chemokine 1)
CYS: Cysteine
DAMP: Damage-associated molecular patterns
DAPI: 4’,6-diamidino-2-phenylindole dihydrochloride
DCs: Dendritic cells
DO: Drop out
DPBS: Dulbecco's phosphate-buffered saline
DSS: Dextran sulfate sodium
xi

EAE: Experimental autoimmune encephalomyelitis
ECL: Enhanced chemiluminescence
ELISA: Enzyme-linked immunosorbent assay
FACS: Fluorescence-activated cell sorting
FAF1: Fas associated factor 1
FBS: Fetal bovine serum
FCAS: Familiar cold autoinflammatory syndrome
FIJI: FIJI is just ImageJ
FLT3-ITD: Fms-like tyrosine kinase 3-internal tandem duplication
FSC: Forward scatter
G-CSF: Granulocyte colony-stimulating factor
GA: Geldanamycin
GAP: Ras GTPase-activating protein
GEO: Gene Expression Omnibus
GLN (Q): Glutamine
GLU (E): Glutamic acid
GLY: Glycine
GM-CSF: Granulocyte-macrophage colony-stimulating factor
GSK3β: Glycogen synthase kinase 3 beta
Hck: Hematopoietic cell kinase
HF: High fidelity
HIS: Histidine
HIV-NEF: Human immunodeficiency virus-negative regulatory factor
xii

HPR: Horseradish peroxidase
HSP: Heat shock protein
IACUC: Institutional Animal Care and Use Committee
IBD: Inflammatory bowel disease
IFNs: Type I interferons
IKK: NF-κB kinase subunit
IL: Interleukin
ILE (I): Isoleucine
IPA: Ingenuity pathway analysis
IRAK1: Interleukin-1 receptor-associated kinase 1
ISG: Interferon stimulated gene
IκB: Inhibitor of κB
KO: Knock out
KRAS: Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
LATS: Large tumor suppressor kinase
LEU (L): Leucine
LPS: Lipopolysaccharides
LRR: Leucine-rich repeat
LTβ: Lymphotoxin beta (a.k.a. TNF-C)
LYS: Lysine
M1: The amino acids in the R1 region were mutated to the amino acids identical to the R1 region
in Lyn
M2: The amino acids in the R2 region were all mutated to Alanine
xiii

M3: The amino acids in the R3 region were all mutated to Alanine
M4: The amino acids in the R4 region were all mutated to Alanine
MDP: Bacterial muramyl dipeptide
MET: Methionine
MHC: Major histocompatibility complex
MIP: Macrophage inflammatory protein
M-MLV: Moloney-murine leukemia virus
mTOR: Mammalian target of rapamycin
MyD88: Myeloid differentiation primary response 88
NBD: Nucleotide-binding domain or nucleotide-binding oligomerization domain (NOD)-like
nucleotide-binding (NBD) domain
NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells
NIK: NF-κB-inducing kinase
Nlrc: Nucleotide-binding oligomerization domain, leucine-rich repeat and CARD containing
protein
Nlrp: Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-
containing protein
NLRs: Nucleotide-binding oligomerization domain-like receptors
p-Tyr: Phosphotyrosine
P2X3: P2X ligand-gated ion channel 3
PAMP: Pathogen-associated molecular patterns
PCR: Polymerase chain reaction
PDB: Protein data bank
xiv

PDT: Pidotimod (3-L-pyroglutamyl-L-thiaziolidine-4-carboxylic acid)
PEP: Phosphoenolpyruvate
PH: Philadelphia chromosome
pHck: Phospho-Hck
PI3K: Phosphoinositide 3-kinases
PKB (a.k.a. AKT): Protein kinase B
PMA: Phorbol 12-myristate 13-acetate
PMNs: Polymorphonuclear leukocytes
PRO: Proline
PRR: Pattern recognition receptor
PTKs: Protein tyrosine kinases
PTP: Protein tyrosine phosphatase
PTPN6: Protein tyrosine phosphatase non-receptor type 6 (a.k.a. SHP-1: Src homology region 2
domain-containing phosphatase-1)
PVDF: Polyvinylidene difluoride
PYD: Pyrin domain
R1, R2, R3, and R4: Designated by us. The four regions in the Hck C1 fragment
RANKL: Receptor activator of nuclear factor kappa-Β ligand
RBC: Red blood cell
RCI: Thermal combined injury
Real time-RCR: RT-PCR
RHD: Rel homology domain
RIPK2: Receptor-interacting serine/threonine-protein kinase 2
xv

RLRs: Retinoic acid-inducible gene-I like receptors
SCFA: Short-chain fatty acid
SD: Synthetic dropout
SD/-Trp-Leu: SD base plus amino acids supplements minus tryptophan and leucine
SD/-Trp-Leu-His-Ade: SD base plus amino acids supplements minus tryptophan, leucine, and
histidine, and adenine
SER (S): Serine
SFKs: Src family of nonreceptor tyrosine kinases
SNPs: Single nucleotide polymorphisms
SSC: Side scatter
STAT5: Signal transducer and activator of transcription 5
STRING: Search tool for recurring instances of neighboring genes
TBST: Tris-buffered saline with Tween 20
TCA: Trichloroacetic acid
TCGA: The Cancer Genome Atlas
TEL: Translocation-Ets-leukemia virus
Thr: Threonine
TLRs: Toll-like receptors
TNF: Tumor necrosis factor
TRAF: Tumor necrosis factor receptor associated factor
TRAF3IP3: TRAF3 interacting protein 3
TRP: Tryptophan
TYR (Y): Tyrosine
xvi

UBA: Ubiquitin-associated domains
UCSF: University of California, San Francisco
Val (V): Valine
WCL: Whole cell lysates
WT: Wild type
X: Stop codon, unknown µl, or unknown amino acid
YPDA: Yeast extract, peptone, dextrose, and adenine hemisulfate

xvii

Acknowledgements

I would like to thank Dr. Curtis Okamoto for his mentoring of me, his generosity for providing
me with funding to support my research, and his flexibility to allow me to explore the science. I
would also like to thank Dr. Honda Alachkar for her mentoring me through the science and the
guidance that she gave me. And, thank you to Dr. Jianming Xie for agreeing to be my committee
member and spending time on my project. Appreciation is also given to Dr. Jae Jung for his
incredible enthusiasm for science, which would always inspire me to pursue the science.

I would also like to thank Dr. Annie Wong-Beringer and the School of Pharmacy for providing
me with funding for the last two years. And, a thank you to Dr. Liana Asatryan and Dr. Daryl L.
Davies to allow me to use the virus culture room. Thanks again to Dr. Liana Asatryan for
showing me how to do virus-cell culture work. I would also like to thank Dr. Julio A. Camarero
and Dr. Wei-Chiang for allowing me to use their cell culture hoods. I want to thank Ms. Meng Li
and Dr. Yibu Chen at the Norris Cancer Hospital for their bioinformatics help.

I also want to thank for Dr. Thomas E. Smithgall for his providing the Hck stable cell lines to us.
Appreciation are also given to Dr. Ian Haworth and Mr. Noam Morningstar-Kywi for their help
on docking analysis. Thank you for Jenny P.-Y. Ting for the conversations of Nlrp12 and Hck in
the 2019 American Association of Immunologists annual meeting.

Thank you to Dr. Lin-chun Chuang for teaching me the yeast two hybrid assay; Dr. Ji-Seung
Yoo for teaching me how to do immunoprecipitations; Dr. Anh Truong and David Tyrpak for
teaching me immunofluorescence; and, Taojian Tu and Dr. Anh Truong for helping me get
xviii

settled in Dr. Okamoto’s lab. I would also like to thank all of the other members in the Jung lab
and the Okamoto lab.

Finally, I would like to thank my college friend, Ms. Biwei Liu. Thank you for always
supporting me through the hard times. And I want to thank for my parents, Xiaoju Xie and
Boyuan Zhang, for their unconditional love and support.

xix

Abstract

The members of the nucleotide-binding oligomerization domain-like receptor (NLR) proteins
family are composed of a N-terminal domain, a central nucleotide-binding domain (NBD), and a
C-terminal ligand-binding domain. They are generally known to be involved in regulation of the
NF-kB signaling pathway. A yeast two-hybrid screen was performed with the cDNA of the pyrin
plus nucleotide binding domain (PYD + NBD) of NLR family pyrin domain-containing12
protein (Nlrp12) as bait and a human leucocyte cDNA library as prey. Hematopoiesis cell kinase
(Hck), a Src non-receptor tyrosine kinase family member was the top hit. Further experiments
confirmed that: 1) the C-terminal 42 amino acids of Hck specifically bound to Nlrp12’s PYD +
NBD, but not to the Nlrp 3 and Nlrp 8, and Nlrp12 PYD + NBD domains; 2) Nlrp12 PYD +
NBD specifically bound to Hck’s C-terminal 42 amino acids but not to those of other src family
kinases; 3) the last 30 amino acids of Hck are sufficient to bind to Nlrp12’s PYD + NBD but not
to Nlrp12’s PYD alone nor to Nlrp12’s NBD alone; and, 4) the last 40 amino acids of
dephosphorylated, but not phosphorylated, Hck preferentially binds to full-length Nlrp12.
Immunoprecipitation experiments showed that Nlrp12 coimmunoprecipitated with Hck when
both were overexpressed in 293T cells. Using the same overexpression system, Nlrp12 and Hck
were co-localized by immunofluorescence. In addition, in THP-1 cells, U937 cells, and RAW
264.7 cells, endogenous Hck co-immunoprecipitated with exogenous, stably expressed Nlrp12.

To examine the functional consequences of Nlrp12 binding to Hck, six different hypotheses were
generated and tested. (1) Since C-terminal Src kinase (Csk) is a tyrosine kinase of the members
of the Src family that specifically binds to the tyrosine negative regulatory residue at the C-
xx

terminal of Src family kinases and tyrosine-phosphorylates them to activate them in mammalian
cells, and Nlrp12 also binds to Hck’s C-terminus, it is hypothesized that Nlrp12 and Csk
compete with each other for binding to Hck’s C-terminus to regulate Hck activity. (2) The
interaction of Nlrp12 with Hck (and possibly tyrosine phosphorylating) causes or prevents
Nlrp12 degradation and/or Hck degradation. (3) Nlrp12 is a p-Tyr substrate of Hck. (4) Hck
modulates Nlrp12-mediated inhibition of the NF-kB pathway, since it has also been shown that
Nlrp12 inhibits the NF-κB pathway. (5) Nlrp12 inhibits or activates Hck. (6) Nlrp12 and Hck
have an impact on proliferation rates in U937 and K562 human leukemia cell lines. However,
none of these hypotheses were supported, or due to time constraints, were able to be tested fully.

Bioinformatic analysis of online databases and typing of blood composition profiles in wild type
and Nlrp12 -/- mice were also performed. Bioinformatic analysis showed that, in some databases,
1) Hck expression is negatively associated with the survival rate of patients with acute myeloid
leukemia (AML), and, in AML patients, 2) Hck co-occured uniquely with Nlrp12, relative to
other Nlrp isoforms, and Hck is co-expressed with Nlrp12. However, no conclusion was reached
concerning the effect of Nlrp12 knocut on the development and progression of AML, as the
number of mice in used in the study was not sufficient.

1
Chapter 1: Nlrp12: a multifunctional protein-effects on immune responses to gut
microbiota

When humans are injured, the body’s the first line of defense is the innate immune system. In
innate immunity, one of the most important defense mechanisms involves the pattern recognition
receptor (PRR), which can recognize two categories of molecules: pathogen-associated
molecular patterns (PAMP) and damage-associated molecular patterns (DAMP) (Takeuchi and
Akira, 2010). PRR comprises four families of proteins: toll-like receptors (TLRs), c-type lectin
receptors (CLRs), retinoic acid-inducible gene- I like receptors (RLRs), and nucleotide-binding
oligomerization domain-like receptors (NLRs, a.k.a. nucleotide-binding oligomerization domain,
leucine-rich repeat receptor) (Takeuchi and Akira, 2010; Wu et al., 2019). The majority of these
genes in the four categories are involved in the upregulation in inflammatory pathways, resulting
in the secretion of proinflammatory cytokines, type I interferons (IFNs), and many other proteins
(Coutermarsh-Ott et al., 2016; Jacobs and Damania, 2012; Takeuchi and Akira, 2010). But some
of these genes are involved in the downregulation in inflammatory pathways, like Nlrp12 (Ye et
al., 2008). TLRs are located in the plasma membrane and endosome system and are known to
defend against infection by bacteria, viruses, protozoa, and parasites (Takeuchi and Akira, 2010).
CLRs are located in the plasma membrane and are known to sense fungal infection (Takeuchi
and Akira, 2010); RLRs are located in the cytoplasm and are known to sense viral infection
(Takeuchi and Akira, 2010). Additionally, NLRs are found in the cytosol and are have been
traditionally understood to sense bacterial infection (Takeuchi and Akira, 2010). However, recent
publications have suggested that some NLR proteins can also sense viral infection (Chen et al.,
2019; Hornick et al., 2018; Sarvestani and McAuley, 2017; Wang et al., 2015).
2
NLRs comprise a family of 22 proteins in humans and 34 proteins in mice (Mason et al., 2012).
All NLRs proteins possess a similar domain structure: an N-terminal pyrin domain, a central
nucleotide-binding domain (NBD, it is also stands for nucleotide-binding oligomerization
domain (NOD)-like nucleotide-binding domain), and a C-terminal leucine-rich repeat (LRR)
domain (Coutermarsh-Ott et al., 2016). The pyrin domain is thought to be responsible for
protein-protein interactions as well as for downstream signaling. The NBD domain binds to
adenosine triphosphate (ATP), which is required for oligomerization of NLRs, and the LRR
domain is responsible for sensing ligands, or of negative regulators or activators of Nlr activation
(Faustin et al., 2007; Kanneganti et al., 2007; Martinon et al., 2004; Sharif et al., 2019). Some
NLRs, such as NLR family pyrin domain-containing protein (Nlrp)1, Nlrp3, NLR family caspase
activation and recruitment domain (CARD)-containing protein 1(Nlrc1) (a.k.a. NOD1), and
Nlrc4, form inflammasomes (Jacobs and Damania, 2012; Sun et al., 2015). Whether Nlrp12 is
involved in inflammasome formation is unclear (Allen et al., 2012; Ataide et al., 2014; Cai et al.,
2016; Silveira et al., 2017; Tuncer et al., 2014; Vladimer et al., 2013; Vladimer et al., 2012).
Nlrp12 is thought to be primarily expressed in dendritic cells, neutrophil, and macrophages, i.e.,
the myeloid lineage, in humans (Chen et al., 2019). In mice, Nlrp12 was previously reported to
be expressed in dendritic cells (DCs) and neutrophils (Arthur et al., 2010; Chen et al., 2019;
Hornick et al., 2018). Additionally, Hornick, et al., also identified Nlrp12 expression in
neutrophils and dendritic cells from the lung (Hornick et al., 2018). Cai et al. (2016) reported that
Nlrp12 is expressed in neutrophils and macrophages in the mouse lung. In addition, some studies
have also reported that Nlrp12 is expressed in alveolar type II epithelial cells in humans and T-
cells (Cai et al., 2016; Gharagozloo et al., 2018; Lukens et al., 2015). The following sections
introduce evidence for Nlrp12’s involvement in NF-κB pathways and their functions, due to its
3
direct relevance in testing Nlrp12’s functional outcomes due to its interaction with Hck.
Nlrp12’s involvement in bacterial, parasitic, and viral infections is not reviewed.

Nlrp12 and regulation of NF-κB pathways
The NLRP12 gene was first identified by Berin’s group and concluded that Nlrp12 regulates
activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway
(Wang et al., 2002). However, later, Ting’s group reported a number of completely different
functions for Nlrp12. They reported that Nlrp12 enhances the RNA and protein expression of
classical and non-classical major histocompatibility complex (MHC) class I gene (Lich et al.,
2007; Williams et al., 2003). Nlrp12 also downregulates both the TLR-and tumor necrosis factor
(TNF)-α-induced canonical NF-κB pathway (Lich et al., 2007; Williams et al., 2005). The same
group also reported that Nlrp12 affected the expression of the non-canonical NF-κB pathway
(Lich et al., 2007).

In the canonical pathway, interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) is
phosphorylated, causing dissociation of IRAK1 from tumor necrosis factor receptor-associated
factor (TRAF) 6. Then TRAF6 is ubiquitinated and thus subject to degradation, and freed
phosphorylated IRAK1 will further interact with downstream molecules and activate the
canonical NF- κB pathway (Iwai et al., 2015; Wertz and Dixit, 2010). Nlrp12 interacts with
IRAK1 and results in a reduction of the phosphorylation of IRAK1, which then dampens its
downstream activation of NF-κB (Williams et al., 2005).

4
In the activated non-canonical NF-κB pathway, TRAF3 is degraded and then releases NF-κB-
inducing kinase (NIK). NIK, as a serine-threonine kinase, can phosphorylate downstream
molecules, resulting in the activation of the non-canonical NF-κB pathway (Lawrence, 2009).
molecules, resulting in the activation of the non-canonical NF-κB pathway (Lawrence, 2009).
When Nlrp12 is present, it performs two roles in the non-canonical pathway. First, it maintains
the level of TRAF3, and second, it inactivates NIK. Nlrp12 binds to with NIK and leads to its
degradation, which results in the inhibition of the NF-κB pathway (Lich and Ting, 2007;
Williams et al., 2005). In addition, Nlrp12 has also been reported to interact with heat shock
protein (HSP) 90, and inhibition of HSP 90 prevents Nlrp12 induced- degradation of NIK
(Arthur et al., 2007). Nlrp12 was also reported to interact with both TRAF3 and NIK. The non-
canonical NF-κB pathway can thus be activated either by TRAF3 degradation and/or NIK
activation. Thus, the non-canonical pathway for NF-κB activation is inhibited (Allen et al.,
2012; Arthur et al., 2007) (Figure 1). Thus, in general, Nlrp12 is thought to be a negative
regulator of NF-κB pathways (Allen et al., 2012; Arthur et al., 2007; Arthur et al., 2010; Silveira
et al., 2017; Wang et al., 2002; Wang et al., 2018; Zaki et al., 2011), although there are reports
that Nlrp12 is also a positive regulator for NF-κB pathways (Wang et al., 2002).

Ye et al. (2008) reported that the Walker A/B domain is a conserved motif that is associated
with phosphate binding. It is located within the NBD domain and specially binds to ATP
resulting in the hydrolysis of ATP. The Walker A/B domain is critical in the function of Nlrp12’s
inhibition of the NF-κB pathway. Cellular expression of Nlrp12 mutations of Walker A/B
resulted in higher expression of proinflammatory cytokines and chemokines than when Nlrp12
5
wild-type (WT) was expressed (Borghini et al., 2011; Ye et al., 2008). However, exactly which
molecules interact with Nlrp12 in effecting this function remains unclear.

Nlrp12 and autoimmune diseases
Autoimmune diseases are a group of diseases characterized by periodic symptoms such as rashes
(Borghini et al., 2011), and there exist numerous mutations in Nlrp12 that may cause
autoimmune diseases. For example, it has been reported that in patient samples, missense
mutations of Nlrp12 will result in periodic fever syndromes (Borghini et al., 2011; Jeru et al.,
2008; Jeru et al., 2011; Xia et al., 2016). Borghini et al (2011) reported a nucleotide 882C-to-G
mutation (at nucleotide 882, C was mutated to G), resulting in the protein Asp294Glu (aspartic
acid (Asp) to glutamic acid (Glu) mutation), resulted in the NF-κB pathway showing no increase
in the p65-induced NF-κB response (Borghini et al., 2011) (Figure 1). Jeru et al. (2008) reported
that two heterozygous nucleotide mutations. These mutations are 1) c.850C>T (at nucleotide
850, C was mutated to T) (p.Arg284X) (at amino acid position 284 of Nlrp12, arginine (Arg) is
mutated to a stop codon (X)) and 2) c.2072 + 3insertT (at intron 3 which is a splice donor site
(splice donor site is the 5’ end of an intron in RNA splicing), T is inserted. This insertion resulted
in the deletion of the upstream last 170 bp of exon 3) resulting in Val635ThrfsX12) (at protein
position 635, valine (Val) was mutated to threonine (Thr). Furthermore, the C to T change and T
insertion also resulted in a frameshift (fs) and generated 11 new amino acids. The longer protein,
now terminating at position 646, had a deleterious effect on NF-κB signaling (Jeru et al., 2008).
Jeru et al (2011) also reported a heterozygous mutation 1054C>T (at nucleotide 1054, C was
mutated to T) resulting in the amino acid mutation Arg352Cys (at amino acid position 352, Arg
was mutated to cysteine (Cys)), but this mutation does not affect NF-κB pathways (Jeru et al.,
6
2011). Xia et al. (2016) detected a nonsense mutation (Trp408X) (at protein position 408,
tryptophan (Trp) is mutated to a stop codon (X)) that is associated with Familiar Cold
Autoinflammatory Syndrome (FCAS). Interestingly, the phenotype of the mutation in FCAS was
observed when a patient was exposed to cold temperatures, and the skin developed a rash.
However, after the patient was exposed to warm temperature, the rash disappeared (Xia et al.,
2016) (Figure 2C). There are many other single nucleotide polymorphisms (SNPs) in Nlrp12
(these can be found on SNPs data resources such as genome ucsc.edu and the dbSNP website),
but whether these are associated with disease is still unknown. Figure 2A and 2B shows the
polymorphisms only that cause diseases.

7

Figure 1: Canonical and non-canonical NF-κB pathways. In the canonical NF-κB pathway,
TLR stimulation will activate canonical NF-κB pathway. Thereafter, myeloid differentiation
primary response 88 (MyD88) is activated, leading to IRAK1 phosphorylation. The IRAK1
photoreaction leads to dissociation of IRAK1 and TRAF6. And subsequently, TRAF6 undergoes
ubiquitinated and degraded. The freed phosphorylated IRAK1 will further phosphorylate NF-κB
kinase subunit (IKK)-γ, leading to IKK-γ activation. Then, inhibitor of κB (IκB) is activated by
phosphorylation, causing the release of p50 and p65 and translocation to the nucleus, where the
complex binds to NF-κB genes (Lawrence, 2009; Rhyasen and Starczynowski, 2014). In the
presence of Nlrp12, the Nlrp12 causes a reduction in IRAK1 phosphorylation. And thus, Nlrp12
dampens the downstream activation of the NF-κB pathway (Lich and Ting, 2007; Williams et al.,
8
2005). In contrast, in the non-canonical NF-κB pathway, cluster of differentiation (CD) 40
activates the non-canonical NF-κB pathway. The activated pathway causes two actions: the
phosphorylation of NIK, and the release of NIK from TRAF3, which is then degraded.
Thereafter, IKK-α dimer is phosphorylated and further activates p100 and RelB by
phosphorylation of p100, resulting in a release of P52 and RelB and their subsequent
translocation to the nucleus. P52 and RelB are transcriptional factors will bind to genes of the
NF-κB pathway (Lawrence, 2009). When Nlrp12 is present, NIK will not be phosphorylated and
TRAF3 will not be degraded. And thus, Nlrp12 prevents downstream activation of NF-κB (Allen
et al., 2012). Picture taken from (Allen, 2014) with modifications.

\

9

Figure 2: Nlrp12 and its polymorphisms. (A). Mutations in the nucleotide sequence of
NLRP12. The mutations in amino acid sequence in Nlrp12, resulting from the mutations of
nucleotide sequences in (A), are shown in parentheses in (B). Data were obtained from genome
ucsc.edu (Kent et al., 2002) and the single nucleotide polymorphism database website
(https://www.ncbi.nlm.nih.gov/snp/) (Sherry et al., 2001) with only mutations noted. Finally, (C)
is an image of a patient whose leg exhibits symptoms of FCAS (Xia et al., 2016).

10
A missense mutation of Nlrp12 also related to atopic dermatitis, a disease characterized as an
intensive immune response to ubiquitous antigens (Macaluso et al., 2007). Macaluso et al (2007)
found that a polymorphism in the 9th intron, Nlrp12_In9 T-‐‑allele (at intron 9 T was found to be a
SNP for C), is significantly associated with atopic dermatitis. The SNP has a weak link to atopic
dermatitis, because the statistical significance was lost after a Bonferroni correction, a type of
statistics test applied to decrease the error of rejecting the null hypothesis. Additionally, it is
unknown why an SNP in the intron causes atopic dermatitis (Macaluso et al., 2007).

Chen et al. (2017) also reported a lower expression of Nlrp12 in patients with adult onset Still’s
disease, an autoinflammatory disease characterized by rashes, fever, arthritis, and multiple other
symptoms. (Chen et al., 2018).

Nlrp12 and autoimmune models
Arthur et al. (2010) showed that the contact hypersensitivity model of Nlrp12 -/- knock out (KO)
mice had less immunological responses compared to WT mice, which is apparently due to less
migration of dendritic cells and less response of dendritic cells and neutrophilia to chemokines
(Arthur et al., 2010). Gharagozloo et al. (2015) reported that Nlrp12 has a protective role in the
experimental autoimmune encephalomyelitis (EAE) model, which is a model for multiple
sclerosis (Gharagozloo et al., 2018), and claimed that Nlrp12 is expressed in myeloid cells
(Gharagozloo et al., 2015). The same group in 2018; however, reported that Nlrp12 is expressed
in T cells of CNS and lymph nodes, mainly CD4+, that will inhibit T-cell proliferation and T-
helper 1 (Th1) response. But at the same time, Nlrp12 can activate the T-cell receptor and
11
prevent activation of NF-κB and protein kinase B (PKB, a.k.a. AKT). They thus concluded that
Nlrp12 exhibited a dual role in the EAE mouse model,

Furthermore, Lukens et al. (2015) also reported that Nlrp12 negatively regulates autoimmune
diseases in the EAE model. They reported that Nlrp12-/- mice developed atypical
neuroinflammatory syndrome, which is caused by Nlrp12-enhanced IL-4 secretion. And they
reported that Nlrp12 is mostly expressed in polymorphonuclear (PMN) leukocytes, CD4+ T-
memory cells, and CD8+ helper T-cells. Moreover, they also found Nlrp12 is a negative
regulator of both the canonical and non-canonical NF-κB pathways (Lukens et al., 2015; Thaiss
and Elinav, 2015). However, further experiments are needed to determine whether Nlrp12 is
expressed in the T-cells as well as exact function Nlrp12 would play in these cells.

Nlrp12 and colitis-induced colon cancer
Liu et al. (2015) analyzed the The Cancer Genome Atlas (TCGA) databases in the Oncomine
platform (http://www.oncomine.org) and reported that Nlrp12 gene expression in patients with
colorectal cancer (CRC) did not significantly differ from those of healthy controls. Additionally,
the level of Nlrp12 gene expression did not differ between various stages of CRC progression.
They also confirmed this finding by analyzing samples from a Chinese patient cohort and found
that there was no difference in Nlrp12 gene expression levels between the healthy controls and
colon cancer patients,, but they reported that Nlrp12 gene expression in CRC patients in the
Gaedcke, Kaiser, and Skrzypczak dataset was significantly higher compared to healthy controls,
while Nlrp12 gene expression in CRC patients in three other databases (Sabates-Belver, Hong,
and TCGA) was not significantly different from that of controls (Liu et al., 2015). However,
12
Allen et al. (2012) reported that in an Nlrp12 -/- mouse model, upon stimulation by dextran
sulfate sodium (DSS), higher inflammation resulted, as measured by the upregulated non-
canonical NF-κB pathway and MAPK pathway and increased levels of the cytokines IL-1 and
IL-1β compared to WT mice. Furthermore, Allen and colleagues also found that Nlrp12 interacts
with TRAF3, which is complementary to the previous findings that Nlrp12 interacts with NIK
(Lich et al., 2007). In the colorectal cancer model where they used Azoxymethane (AOM) +
DDS, they found that 1) Nlrp12 attenuated tumorigenesis; 2) Nlrp12’s attenuation of
tumorigenesis occurs in both hematopoietic and nonhematopoietic compartments; and, 3) Nlrp12
attenuation of tumorigenesis resulted in a decrease of non-NF-κB-induced cytokine expression
and expression of chemokines (the chemokine (C-X-C motif) ligand (CXCL) 12 and CXCL13)
in mouse colon tissues (Allen et al., 2012). Interestingly, Zaki et al. (2011) independently
similarly reported that Nlrp12-deficient mice are more susceptible to colitis and colitis-induced
tumorigenesis. Zaki, et al., (Zaki et al., 2011) showed that Nlrp12 is responsible for decreasing
the secretion of cytokines L-1β, IL-6, TNF-α, IL-17, and IL-15 and decreasing of the
chemokines granulocyte colony-stimulating factor (G-CSF), C-C motif chemokine ligand 11
(CCL11, a.k.a. eotaxin), CXCL-1 (a.k.a. KC), CXCL10 (a.k.a. IP-10), macrophage inflammatory
protein (MIP)-1α, MIP-1β, and MIP2. However, they said that only the hematopoietic
compartment was responsible for tumorigenesis and that Nlrp12 negatively regulates the
canonical NF-κB pathway without mentioning the non-canonical NF-κB pathway.

Nlrp12’s role in regulating gut microbiota
Recently, Nlrp12’s role in the regulation of gut microbiota has been reported. Chen, et al., (2017)
found that Nlrp12 is lower in patients with ulcerative colitis. These patients were twins, one of
13
whom had colitis, while the other was a healthy control. In addition, they used Nlrp12 -/- mice
which showed less diversified microbiomes and a different bacterial composition compared to
WT mice. And there is a great dissimilarly between Nlrp12 -/- mice and WT mice than within
Nrlp12 mice or WT mice. Furthermore, they also showed that the amounts of gut microbiota of
the order Bacteroidales and Chostridiales, and the family of Lachnospiraceae are lower, and the
amount of bacteria of the family of Erysipelotrichaceae is higher in Nlrp12 -/- mice.
Subsequently, they co-housed Nlrp12 -/- mice and WT mice and found that in Nlrp12 -/-, colitis
induced by DDS was attenuated; they also transferred microbiota from Nlrp12 -/- mice to WT,
and found that inflammation increased under colitis-induced cancer. These two experiments
indicate that the microbiota can help decrease inflammation and DDS induced cancer.
Furthermore, when the mice were given Lachnospiraceae bacteria directly, or TNF-α and IL-6,
which are known to be a treatment for inflammatory bowel disease (IBD), they found that
Nlrp12 -/- mice recovered from colitis and dysbiosis, indicating that Nlrp12 -/- mice had less
diversity of microbiomes compared to WT mice. (Chen et al., 2017; Prochnicki and Latz, 2017).
Moreover, Truax et al. (2018) connected gut microbiota to obesity by demonstrating that Nlrp12
in myeloid cells maintains body weight by inhibiting the excess inflammatory response induced
by high fat diet. In the condition where a high fat diet was fed to mice, Nlrp12 inhibited
inflammation. However, when Nlrp12 was not present, inflammation was higher, and thus,
cytokines, including IL-1β, IL6, TNF, and anti-microbial peptides were higher. More
importantly, Lachnospiraceae decreased and Erysipelotrichaceae increased in Nlrp12 -/- mice
(Truax et al., 2018). These microbiomes normally induce short-chain fatty acid (SCFA)
synthesis, which will create negative feedback on inflammation. But when Nlrp12 is not present,
these microbiomes are in dysbiosis, and thus cannot produce sufficient SCFA. Thus, the negative
14
feedback loop in inhibited. Therefore, weight gain, adipogenesis, insulin tolerance, increased
inflammation in fat, and macrophage infiltration in fat will occur. Truax et al. (2018) have also
used Lachnospiraceae (can produced SCFA) to treat WT and Nlrp12-/- mice and found that
body weight gain and inflammation were attenuated more in Nlrp 12-/- mice in response to high-
fat diet, while in WT mice these effects were less obvious (Prochnicki and Latz, 2017; Truax et
al., 2018).

Nlrp12 and bone physiology
Krauss et al. (2015)’s finding shed light on a new role of Nlrp12 outside of inflammation-related
diseases, that is, Nlrp12 can decrease the development of osteoclasts by its suppression of the
non-canonical NF-κB pathway in osteoclast precursors, and expression of Nlrp12 results in the
downregulation of receptor activator of nuclear factor kappa-Β ligand (RANKL) which is known
to be a ligand that activates both the canonical and non-canonical NF-κB pathways (Krauss et al.,
2015). They thus characterized a new pathway in which Nlrp12 is involved. In this pathway
Nlrp12 inhibits RANKL, and RANKL promotes NF-κB activation in the osteoclast precursors,
which promotes osteoclast differentiation.

Nlrp12 is correlated with prostate cancer
Karen et al. (2017) through the Gene Expression Omnibus (GEO) public datasets, found that
Nlrp12’s expression level was significantly higher in malignant prostate tissues compared to the
adjacent normal tissues in prostate cancer patients (Karan et al., 2017). However, this is only a
correlation study, and whether Nlrp12 has a role in prostate cancer is unknown.

15
Nlrp12 in other functional roles
The following data are only correlations. It has been reported that Nlrp12 mutations, i.e.,
c.3049C>G (at nucleotide 3049, C was mutated to G) resulting in the amino acid mutation
Arg1017Gly (at protein position 1017, Arg was mutated to glycine (Gly)) and c. 3042-
3043delAG (at nucleotide 3042 to 3043, the A and G were deleted) giving rise to the mutation
Gly1014fs, which is a frame shift mutation at amino acid 1014. This mutation was significantly
associated with preterm birth, suggesting that innate immunity may be an etiologic factor in
spontaneous preterm birth, and PAMP and DAMP could play a role in preterm births (Modi et
al., 2017; Strauss et al., 2018).

Marzano et al. (2017), through whole-genome sequencing, have reported that the Nlrp12
mutation Phe402Leu (at protein position 402, phenylalanine (Phe) was mutated to leucine (Leu))
was associated with pyoderma gangrenosum, which is an ulcer-related skin disease, with acne
and suppurative hidradenitis (Marzano et al., 2017). Van Schouvenburg, et al., (2015), found
Nlrp12 variants, i.e., c.910C>T (at nucleotide 910, C was mutated to T) resulting in the amino
acid mutation His304Tyr (at protein position 304, histidine (His) was mutated to tyrosine (Tyr)),
and c.1148C>G (at nucleotide 1148, C was mutated to G) resulting in the amino acid mutation
Thr406Arg (at protein position 406, Thr was mutated to Arg), was associated with common
variable immunodeficiency disorders (van Schouwenburg et al., 2015).

Other data have come from functional studies. Linz et al. (2017) have reported that Nlrp12 is
highly expressed in the thermal combined injury (RCI) model; the mechanism being that Nlrp12
acts as a checkpoint of TNF and also prevents hematopoietic apoptosis (Linz et al., 2017).
16
Cecrdlova et al. (2016) have reported that Nlrp12 is upregulated at 4 hours when the THP-1 cells
were treated with manumycin A, which is an inhibitor of farnesyltransferase derived from
Streptomyces (Cecrdlova et al., 2016). Fogli et al. (2014) have reported that pidotimod (3-L-
pyroglutamyl-L-thiaziolidine-4-carboxylic acid) (PDT), which is a peptide used to treat
infections, upregulated Nlrp12 in MM6 cells in the human monocytic cell line (Fogli et al.,
2014).

Conclusion
Nlrp12 is a protein with multifaceted functions. Clearly, there are still many potential functions
that have not been mechanistically characterized or the published findings are contradictory. For
example, whether Nlrp12 is involved in inflammasome formation remains unclear. Also, it is not
known if the mechanisms of all of the functions of Nlrp12 simply involve Nlrp12 inhibiting NF-
κB pathway, and further testing is needed to determine its role in this process.

17
Chapter 2: Hematopoietic cell kinase (Hck)’s structure and functions

Hck structure
Hck belongs to the Src family of non-receptor tyrosine kinases, of which there are 9 family
members, Src, Yes, Fyn, Lck, Fgr, Blk, Lyn, Yrk, and Hck (Kim et al., 2009). Src, Yes, and Fyn
are ubiquitously expressed, while Lck, Hck, Blk, Fgr, and Lyn are primarily expressed in
hematopoietic cells (Martin, 2001). The primary amino acid sequences of these family members
are very well conserved. They share a conserved SH2 domain, which is a domain that recognizes
the phosphoserine peptide; an SH3 domain, which is a domain that recognizes proline-rich
domains; an SH1 domain, which is the kinase domain, and a C-terminal tail, which contains an
autoinhibitory phosphorylation site (Boggon and Eck, 2004). The uniqueness of each family
member is the SH4 domain (Figure 3). As shown in Figure 3, there are also two linkers known to
connect with SH3 and SH2 domains (SH3-SH2 linker) and SH2 and SH1 domains (SH2-SH1
linker). Hck’s crystal structure (in its inactive form) was solved in 1997 (Sicheri et al., 1997) and
the protein structure from protein data bank (PDB) website (https://www.rcsb.org) (accession
number: 1AD5) was downloaded for the following experiments in chapter 4 (Figure 4).
Subsequently, other studies have reported Hck’s crystal structure with a Src-family kinase
inhibitor (Alvarado et al., 2014; Sicheri et al., 1997).

There are four total isoforms in Homo sapiens, two of the isoforms are p61 and p59 (The UniPort
Consortium, 2019). P61 and p59 share the same transcript; P61 uses an uncommon start codon
CTG, and p59 uses the common one, ATG (Robbins et al., 1995). The biochemical differences
of the two isoforms are: 1) P61 has a myristylation site on the glycine at the +2 position
18
(sequences from pubmed isoform p61), and P59 has a myristylation site on glycine at +2
position, plus a palmitoylation site at serine at the +6 position (sequences from pubmed isoform
p59) (Poh et al., 2015). And, 2) P61 is mainly found on lysosomes (Poh et al., 2015) and P59 is
distributed mainly in the plasma membrane (Poh et al., 2015). There are two tyrosine
phosphorylation sites on Hck. One site is the positive regulator of Hck, Tyr410 in the SH1
domain, the other site is the negative regulator of Hck, Tyr521, in the C-terminal tail (sequence
from the pubmed Hck p61, accession number: AAH94847.2) (Musumeci et al., 2015).

Hck activity is strictly regulated in cells, and Hck is usually maintained in its inactive status.
Inactive Hck status is maintained by the phosphorylation of Tyr521, which binds it
intramolecularly to the SH2 domain, and also by SH1-SH2 linker, which is connected to SH3
domain (Figure 4). The activation of Hck is mediated by dephosphorylation of Tyr521 by CD45
(or protein tyrosine phosphatase receptor type C), protein tyrosine phosphatase non-receptor type
6 (PTPN6) (a.k.a. Src homology region 2 domain-containing phosphatase-1 (SHP-1)), protein
tyrosine phosphatase (PTP) 1B, and PTPa, and other PTPases (Poh et al., 2015). Hck can also be
activated by autophosphorylation of Tyr410 (Poh et al., 2015). On the other hand, inhibition of
Hck is mediated by the C-terminal Src kinase (Csk) family of tyrosine protein kinases: Csk and
checkpoint kinase (CHK) phosphorylation of Tyr521. The inhibition of Hck can also occur when
CD45, SHP1, T-cell PTP, and phosphoenolpyruvate (PEP) dephosphorylation of Tyr410 (Poh et
al., 2015). In contrast, Hck activation can also be achieved by overexpression, treatment of cells
with Lps, IL-2, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and by
interaction with human immunodeficiency virus-negative regulatory factor (HIV-NEF), which is
a protein that is the ligand of SH3 domain and thus binds tightly to the SH3 domain of Hck
19
(Moarefi et al., 1997). The NEF has PxxP motif that binds to SH3 domian, which causes
displacement of the SH3-SH2 linker, and further results in a conformational change that causes
Hck activivation (Adams, 2003; Moarefi et al., 1997). Additionally, activation does not require
the C-terminal tail dephophorylatation, which suggested a different machenism of Hck activation
(Lerner and Smithgall, 2002).

Hck’s functions
Hck is abundantly expressed in monocyte and macrophage (Normand et al.; Quintrell et al.,
1987; Ziegler et al., 1987). Since the members of the Src-family kinases are generally highly
conserved and abundant, likely due to functional redundancy by other Src-family members, only
the simultaneous knock out of two or three Src family kinases knock out mice will bear some
phenotype. For example, Lck/Hck/Fgr triple knock out mice showed dampened anti-virus
response (Liu et al., 2017). And Hck/Lyn/Fgr triple knock out bone marrow derived cells
induced the chronic myeloid leukemia (CML) after the cells are transduced with breakpoint
cluster region protein/ Abelson murine leukemia (Bcr/ABl) 1 (Hu et al., 2004). However, Hck-/-
mice generally do not appear to have a phenotype even though Hck has many functions. On the
other hand, Hck Y499F, in which the tyrosine residue at the inhibitory regulatory site is mutated
to phenylalanine, is a constitutively activated form of Hck. When expressed in transgenic mice,
these mice developed a colitis-induced colon cancer (Poh et al., 2017), similar to that in Nlrp12 -
/- mice.

Hck’s function in leukemia
A more complete review of how Hck affects leukemia can be found in (Poh et al., 2015). Briefly,
Hck is found highly expressed in CML, multiple myeloma, and acute lymphoblastic leukemia.
20
The functions related to leukemia is Hck cause the high proliferation, except that Hck in the
CML is known to cause the cell transformation (Poh et al., 2015).

Lopez et al. (2016) reported that the Hck pathway may be involved in acute myeloid leukemia
(AML) cell lines (MV4-11, MOLM-14, and TF-1) and primary murine hematopoietic cells. Hck
is downstream of feline McDonough sarcoma (fms)-like tyrosine kinase 3 (FLT3)-internal
tandem duplication (FLT3-ITD). FLT3 is a kinase. ITD mutation is the most common mutation
for FLT3. ITD, as its name, is the in-frame internal tandem duplications and will result in the
constitutive activation of FLT3. This will result in Hck activation. And Hck activation will
induce the cyclin-dependent kinase 6 (CDK6) activation. CDK6, is a protein that controls the cell
cycle. Thus the mutation of FLT3 will ultimately result in uncontrolled proliferation of the cells
(Lopez et al., 2016). Roversi et al. (2014) also reported that Hck plays a role in myelodysplastic
syndromes and acute leukemia. They found that phosphorylation of AKT and mammalian target
of rapamycin (mTOR) is decreased when Hck is knock down. This indicate that Hck can activate
phosphoinositide 3-kinases (PI3K) pathway that resulted in a higher rate of cell survival and
proliferation (Roversi et al., 2014).

In addition to FLT3/ITD mutation, there are two other mutations caused by fusions in leukemia
that are associated with Hck. These two mutations are the BCR/ABL and translocation-Ets-
leukemia virus (TEL)/ABL. It has been reported that the BCR/ABL interacts with Hck, and leads
to Hck activation. Active Hck phosphorylated signal transducer and activator of transcription 5
(STAT5). This BCR/ABL-Hck-STAT5 is responsible for the transformation of myeloid cells in
AML or CML (Klejman et al., 2002; Lionberger et al., 2000; Warmuth et al., 1997). For the
21
TEL/ABL, it is reported that Hck is highly phosphorylated in the Ba/F3 cell that expressed
TEL/ABl (Pecquet et al., 2007).

Additionally, Hck is also reported to be negatively associated with leukemia. Hoshino et al.
(2007) showed the Hck gene to be highly methylated in Bcr-Abl negative leukemia (Hoshino et
al., 2007). And methylation is reduced in Hck mRNA and protein expression. So Hck may be
considered as a tumor suppressor in Bcr-Abl negative leukemia. Additionally, Hck is also found
to be reduced in AML with Philadelphia chromosome (Ph) negative patients and acute
promyelocytic leukemia (APL) cases (Poh et al., 2015).

Hck and inhibitors related to leukemia
There are publications reporting several inhibitors of Hck that can inhibit leukemia. First,
Roversi et al. (2017) reported that they discovered a Hck inhibitor, iHCK-37, which caused
leukemic cell death (lukemia cell lines, i.e., HEL, HL60, KG1a, K562 and U937 cells) but did
not affect normal hematopoietic stem cells (CD34+ cells, i.e., hematopoietic stem and
progenitor cells). They also found that Hck regultes the differentiation of erythroid cells and
additionally reported that Hck levels were high in the stem cells in patients with myelodysplastic
syndromes and AML. Thus the inhibitor can target these diseases which are related to dieased
erythropoiesis. In additon, Hck is related to PI3K and MAPK pathways, which are also inhibited
by iHCK-37. Inerestingly, Roversi’s group found K562 has high level of Hck, while others have
reproted that Hck has no expression in K562 (Roversi et al., 2017; (Pene-Dumitrescu et al.,
2008; Pene-Dumitrescu and Smithgall, 2010). Secondly, Saito et al. (2013) found another Hck
inhibitor, RK-20449, that can bind to the active site of Hck to inhibit its activity. They also
reported that Hck is highly expressed in leukemia stem cells compared with hematopoietic stem
22
cells. Furthermore, RK-20449 can inhibit Hck in the leukemia stem cells that will cause
chemotherapy resistant, and thus can prevent the AML relapse (Saito et al., 2013). Third, Patel et
al. (2017) also reported a Hck inhibitor, A-419259, that binds to the active site of Hck and is a
broad-spectrum Src kinase inhibitor, can keep Hck expression low in FLT3/ITD positive AML
cell lines (Patel et al., 2017). It has also been reported that A-419259 can induce apoptosis and
prevent proliferation in the CML cell lines (K562 cell line that were stably expressed with Hck
and human TF-1 cell line that were stably expressed with Bcr-Abl and Hck) (Pene-Dumitrescu et
al., 2008). Forth, Naganna et al. (2019) developed an alkynyl aminoisoquinoline and alkynyl
aminonaphthyridine compound. They showed that this compound can inhibit FLT3 and also Src
family kinases, which are downstream of FLT3 kinase, using in vitro and in vivo models.
Therefore, these Flt3-Src dual kinase inhibitors can inhibit AML proliferation (Naganna et al.,
2019). Lastly, Hu et al (2004) reported that inhibition of Lyn, Hck, and Fgr kinase using a kinase
inhibitor CGP76030 in the B-lymphoid cells that expressed Bcr-Abl can inhibit the proliferation
of B-lymphoid cells, and thus can inhibit Ph-positive acute lymphoblastic leukemias (ALL).
They also reported that combination of CGP76030 and imatinib resulted in better outcome (cell
apoptosis and survival rate of the mice) than just used CGP76030 or imatinib alone (Hu et al.,
2004).
These studies demonstrate that Hck may be an important kinase in leukemias and many Hck
inhibitors are under development to treat leukemias. However, whether these inhibitors have
effects on other Src non-receptor tyrosine kinases are questionable. Further study is needed to
clarify whether these inhibitors would impact other cancers that Hck induced.

23

Figure 3: Two isoforms of Hck and schematic drawing of the structure of Hck. The two
isoforms of Hck share the same transcript but different start codons. P61 isoform used the start
codon CTG. P59 isoforms used the start codon AUG. Hck is divided by four different domains,
SH1 domain, which is the kinase domain; SH2 domain, which is responsible for the
phosphoserine peptide binding; SH3 domain, which is responsible for proline-rich domain
binding; and a unique domain, which is unique among all the Src family proteins of non-receptor
tyrosine kinases.

81 140 240 262 1 150 526

SH3 SH2 SH1 Unique
514
1 505

SH3 SH2 SH1 Unique
P61 Hck
P59 Hck
24

Figure 4: Hck crystal structure (picture derived from pMOL software) (protein data bank
(PDB) accession number is 1AD5). This image shows the inactive status of Hck. In the inactive
status, C terminal is phosphorylated at Tyr521 site, which binds to SH2 domain, and SH1-SH2
linker (showed by green color in the graph) is connected to SH3 domain. This resulted in a
confirmation that the phosphorylation active site (Tyr410) is masked, and thus Hck cannot be
active by autophosphorylation of Tyr410 site.

25
Chapter 3: Material and Methods

Reagents and cells
U937, THP-1, RAW 264.7, and 293T cells were all purchased from American Type Culture
Collection (ATCC) (Manassas, VA). pCDNA-Nlrp12 plasmid and pCDNA-Nlrp3 plasmid were
gifts from Dr. Jenny P. Y. Ting (University of North Carolina-Chapel Hill). pCDNA-Nlrp8
plasmid was purchased from Genescript (Piscataway, NJ). Hck p61 was obtained from yeast
two-hybrid. Csk was purchased from Addgene (Watertown, MA). pCDNA-Nlrp1, pCDNA-
Nlrp2, pCDNA-Nlrp5, pCDNA-Nlrp6, pCDNA-Nlrp7, pCDNA-Nlrp9, pCDNA-Nlrp10,
pCDNA-Nlrp11, pCDNA-Nlrp13, and pCDNA-Nlrp14 were transferred from a post-doctoral
fellow in Jae Jung’s lab.

All other chemicals were reagent grade. The mouse monoclonal anti-FLAG antibody, the rabbit
monoclonal anti-FLAG antibody, and the mouse monoclonal anti-GAPDH antibody, and the
mouse monoclonal anti- β actin antibody were purchased from Sigma-Aldrich (St. Louis, MO).
The mouse monoclonal anti-Hck antibody, the mouse monoclonal anti-Csk antibody, the mouse
monoclonal anti-TRAF3 interacting protein 3 (TRAF3IP3) antibody, and the mouse IgGκ
binding protein-horseradish peroxidase (HRP) for detecting the anti-Hck antibody were
purchased from Santa Cruz Biotechnology (Dallas, TX). The mouse monoclonal anti-V5
antibody, the rabbit monoclonal anti-V5 antibody, the mouse monoclonal anti-phosphotyrosine
(p-Tyr)-100 antibody, the rabbit polyclonal anti-p-Y416 Src, the rabbit polyclonal anti-p-Tyr521
Src antibody, the mouse monoclonal anti-phospho-IκB-α antibody, the rabbit polyclonal anti-
IκB-α antibody, the polyclonal rabbit HRP-conjugated anti-mouse IgG secondary antibody, the
26
goat anti-rabbit IgG HRP-linked secondary antibody, rabbit anti-mouse IgG HRP-linked
secondary antibody, and anti-mouse IgG isotype control were purchased from Cell Signaling
(Beverly, MA). The mouse monoclonal 4G10 anti-phosphotyrosine antibody was purchased
from EMD Millipore (Temecula, CA). The Clean-Blot™ IP Detection Reagent, goat anti-mouse
IgG (H+L) cross-adsorbed secondary antibody, conjugated to Alexa Fluor 488, goat anti-rabbit
IgG (H+L) cross-adsorbed secondary antibody, conjugated to Alexa Fluor 555. 4’,6-diamidino-
2-phenylindole dihydrochloride (DAPI), and Lipofectamine® 3000 were purchased from
Thermo Fisher Scientific (Grant Island, NY).

Cloning
Nlrp12’s PYD + NBD (a.a. 1-531) was amplified by polymerase chain reaction (PCR) (primers
see Table 1), from a pCDNA-Nlrp12 plasmid, was digested with enzymes NdeI and SalI, and
subcloned into the pGBKT7 vector (Takara). The sequence encoding the PYD + NBD of Nlrp3
(a.a. 1-536) was amplified by PCR (see table 1 for primers), digested with enzymes NdeI and
BamHI, and subcloned in the pGBKT7 vector. The sequence encoding the PYD + NBD of Nlrp8
(a.a. 1-527) was amplified by PCR (see Table 1 for primers), digested with enzymes EcoRI and
BamHI, and subcloned into the pGBKT7 vector. The entire sequences of the C-terminal 42
amino acids of Fgr, Src, Yes, and Fyn, and 40 amino acids of Blk, Lck, Lyn, and Hck (The C
terminal 40 (or 42) amino acids is called “C1”), and Hck constructs C2, C3, C4, C5, C6, C7, N1,
N2, N3, N4, N5, M1, M2, M3, M4, and Hck C1 mutations for alanine scanning with F503A,
E504A, Y505A, I506A, Q507A, S508A, V509A, L510A, and D511A, were all synthesized from
IDT DNA (Coralville, IA) and cloned into the pACT2 vector (Takara).

27
pEFIRES-P-puro and pEFIRES-P-hygro vectors (Hobbs et al., 1998) were obtained from Dr. Jae
Jung’s lab, and depending on our needs, 3FLAG- and V5-epitope tags were added later into the
pEFIRES-P-puro and pEFIRES-P-hygro vectors. The peptide sequence of the 3FLAG epitope is
DYKDDDDKDYKDDDDKDYKDDDDK. The peptide sequence of the V5 epitope is
GKPIPNPLLGLDST. The multiple cloning sites of pEFIRES-P-puro and pEFIRES-P-hygro
were modified in Jae Jung’s lab.

The cDNAs for Nlrp1, Nlrp2, Nlrp3, Nlrp7, Nlrp8, and Nlrp10 were amplified by PCR (primers
see table 1), from the vectors pCDNA-Nlrp1, pCDNA-Nlrp2, pCDNA-Nlrp3, pCDNA-Nlrp7,
pCDNA-Nlrp8, and pCDNA-Nlrp10, individually, digested with enzymes AflII and XhoI, and
subcloned into the pEFIRES-P-puro-3FLAG vector. The cDNAs from Nlrp5 and Nlrp6 were
amplified by PCR (see Table 1 for primers), from the vectors pCDNA-Nlrp5 and pCDNA-Nlrp6,
individually, digested with AflII and XbaI, and subcloned into pEFIRES-P-puro-3FLAG vector.
The cDNA from Nlrp12 was amplified by PCR (see Table 1 for primers), from the vector
pCDNA-Nlrp12, digested with AflII and XbaI, and subcloned into pEFIRES-P-puro-3FLAG and
pEFIRES-P-puro-V5 vectors. The cDNAs for Nlrp9, Nlrp13, and Nlrp14 were amplified by PCR
(primers see table 1), from the vectors pCDNA-Nlrp9, pCDNA-Nlrp13, and pCDNA-Nlrp14,
individually, digested with enzymes NdeI and XhoI, and subcloned into the pEFIRES-P-puro-
3FLAG vector. The cDNA for Nlrp11 was amplified by PCR (primers see table 1), from the
vector pCDNA-Nlrp11, digested with enzymes NdeI and EcoRI, and subcloned into the
pEFIRES-P-puro-3FLAG vector. Sequences encoding Hck p61 and p59 isoforms were amplified
by PCR (see Table 1 for primers), from the vector pACT2-Hck p61 (from yeast two-hybrid),
digested with AflII and XbaI, and subcloned into pEFIRES-P- hygro vector. The cDNA for Csk
28
was amplified by PCR (primers see Table 1), from the vector pcFLAG-Csk-WT, digested with
enzymes AflII and XbaI, and subcloned into the pEFIRES-P-puro vector. Final plasmids were
sequenced to confirm the constructs.

29
Table 1: Primers used in cloning (The underline shows the restriction enzymes)
Name Primers
Nlrp12 5’ primer
(cloned into PGBKT7)
GATCTACATATGCTACGAACCGCAGGCAG
Nlrp12 3’ primer
(cloned into PGBKT7)
GATGTAGTCGACTCACCCCTCGTCCAGGATATAGTACATAGCT
Nlrp3 5’ primer
(cloned into pGBKT7)
GATCTACATATGAAGATGGCAAGCACCCGCTGCA
Nlrp3 3’ primer
(cloned into PGBKT7)
GATGTAGGATCCTCACAGCAGGTAGTACATGGCGGCAAAGA
Nlrp8 5’ primer
(cloned into pGBKT7)
GATCTAGAATTCATGAGTGACGTGAATCCACCCTCTG
Nlrp8 3’ primer
(cloned into PGBKT7)
GATGTAGGATCCTCAGAGTCTTTGTGGGAAACAGAGAACA
Hck p61 5’ (cloned
into pEFIRES-P-
hygro)
GATCTACTTAAGGCCACCATGGGGGGGCGCTCAAGCTGCGAGG
Hck p59 5’ (cloned
into pEFIRES-P-
hygro)
GATCTACTCGAGGCCACCATGGGGTGCATGAAGTCCAAGTTCC
30
Hck p61/p59 3’
(cloned into
pEFIRES-P-hygro)
GTCGATTCTAGATCATGGCTGCTGTTGGTACTGGCTC
Csk 5’ (cloned into
pEFIRES-P-puro)
GATCTACTTAAGGCCACCATGTCAGC AATACAGGCCGCCTGGC

Csk 3’ (cloned into
pEFIRES-P-puro)
GTCGATTCTAGATCACAGGTGCAGCTCGTGGGTTTTG
Nlrp1 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGGCTGGCGGAGCCTGG
GGCCGCC

Nlrp1 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGGCTGCTGAGTGGCAGGAGTCCCTTT
Nlrp2 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGATGGTGTCTTCGGCGCAGATGG

Nlrp2 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGTCAGATCATGAAGTCATGAGAAGAA
Nlrp3 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGAAGATGGCAAGCACCCGCTGCA

31
Nlrp3 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGCCAAGAAGGCTCAAAGACGACGGTC

Nlrp5 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGAAGGTTGCAGGAGGACTTGAAC

Nlrp5 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATTCTAGAGTTTTTCCACCAGTACCGGTCATCT
Nlrp6 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGGACCAGCCAGAGGCCCCCTGCT

Nlrp6 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATTCTAGAGAAGGTCGAGATGAGTTCCTTGGGA
Nlrp7 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGACATCGCCCCAGCTAGAGTGGA

Nlrp7 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGGCAAAAAAAGTCACAGCACGGAGGT
32
Nlrp8 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGAGTGACGTGAATCCACCCTCTG

Nlrp8 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGAGGATTAATCTGGGATAGGCAGTCA
Nlrp9 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTAGCTAGCGCCACCATGGCAGAATCTTTTTTTTCGGATT

Nlrp9 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGGAGGAGCACACCCCTGATCTTGTAT
Nlrp10 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTACTTAAGGCCACCATGGCCATGGCCAAGGCCAGAAAGC

Nlrp10 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGTATGTAAGTATTTTTTGGTGTTTCC
Nlrp11 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTAGCTAGCGCCACCATGGCAGAATCGGATTCTACTGACT

33
Nlrp11 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATGAATTCAAGGGGTTGCCTAGATGCTGTATTT
Nlrp12 5’ (cloned into
pEFIRES-P-puro-
3FLAG and
pEFIRES-P-puro-V5)
GTCCAGCTTAAGATGCTACGAACCGCAGGCAG
Nlrp12 3’ (cloned into
PEFIRES-P-puro-
3FLAG and
pEFIRES-P-puro-V5)
CTGAATTCTAGAGCAGCCAATGTCCAAATAAGG

Nlrp13 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGCCCGAGTTTCTGCAGCCTGCATGTC
Nlrp14 5’ (cloned into
pEFIRES-P-puro-
3FLAG)
GATCTAGCTAGCGCCACCATGGCAGATTCATCATCATCTTCTT

Nlrp14 3’ (cloned into
pEFIRES-P-puro-
3FLAG)
GTCGATCTCGAGGAAACACCACCACCAAGACACATCT

34
PCR
PCR was performed following the protocol for Phusion® high-fidelity PCR master mix (Thermo
Fisher Scientific). Briefly, 25 µl of 2 x Phusion® master mix with high fidelity (HF) buffer, 1 µl
forward primer, 1 µl reverse primer, 2.5 µl DNA, 1.5 µl of DMSO, and 19 µl of UltraPure
TM

DNase/RNase-free distilled water (Thermo Fisher Scientific) were added into one tube. After
mixing well, the tube was put in a PCR machine with the following cycles: 98 °C for 30 s, 35
cycles of 98 °C for 10 s and 72 °C for 30s/kb, 72 °C for 30 min, and 4 °C for holding.

Yeast two-hybrid assay
Yeast transformed with the plasmid PGBKT7-Nlrp12’s PYD + NBD
Yeast strain AH109 (Takara, Mountain View, CA) was stored frozen at -80°C. Upon use, it was
streaked onto an agar plate supplemented with yeast extract, peptone, dextrose, and adenine
hemisulfate (YPDA) (Takara), which was then placed in a 30°C incubator overnight. The
growing colonies were picked and placed into 10 ml of YPDA media, incubated overnight with
shaking at 30°C. Then they were transformed with Nlrp12’s PYD + NBD plasmid, following
recommendations in the Takara manual (Matchmaker
TM
Gal4 Two-hybrid System 3 and
Libraries User Manual). A 4ml of 1 M of 3-amino-1,2,4-triazole (3-AT) was added into 800 mL
of the YPDA medium that was used for making the plates due to a leakage problem. 3-AT is a
competitive inhibitor for the product of the HIS3 reporter gene. Yeast cells were lysed using a
trichloroacetic acid (TCA) method, and western blots were performed to check Nlrp12’s PYD +
NBD protein expression (data not shown).

35
Library scale transformation of cDNA library into yeast that have been transformed with the
PGBKT7-Nlrp12 PYD + NBD plasmid
A human leukocyte Matchmaker
TM
cDNA library was purchased from Takara. The library was
titered according to the Matchmaker
TM
Gal4 Two-hybrid System 3 and Libraries User Manual.
The colony-forming units (CFU) for the library were calculated to be about 3.1 x 10
9
CFU/ml.
Four large scale transformations (equal to one library scale) were done at the same time
following the Matchmaker
TM
Gal4 Two-hybrid System 3 and Libraries User’s Manual. To
calculate the efficiency of yeast two-hybrid (i.e., the number of colons obtained per µg of library
plasmid DNA) and to check the expressions of “bait” and “prey” plasmids, small amounts of the
solution, diluted 10X, 100X, 1000X, and 10,000X were plated in 2DO (drop out) plates, i.e.,
synthetic dropout (SD)) plus amino acids supplements minus tryptophan and leucine (-Trp-Leu).
The yeast two-hybrid efficiency obtained was 3 x 10
6
CFU per µg of library plasmid DNA. In
addition, if the yeast grew on the 4DO plates, i.e., SD plus amino acids supplements minus
tryptophan, leucine, histidine, and adenine (-Trp-Leu-His-Ade) + 3-AT plates, the “bait” and
“prey” plasmids were considered to be interacting with each other.

Small scale transformation
The yeast two-hybrid co-transformation on a small scale for all of the direct screenings was
performed following the small-scale transformation protocol in the Matchmaker
TM
Gal4 Two-
hybrid System 3 & Libraries User’s Manual. The direct screens were to test whether Nlrp12
PYD + NBD and Hck specifically interact; identification of Hck Phe503, Glutamine (Gln) 507,
Leu510, and Asp511 being critical for binding with Nlrp12 PYD + NBD; and the
characterization of the Hck C-terminal 30 amino acid fragment binding to Nlrp12 PYD + NBD.
36
Docking analysis
The structure of Nlrp12’s PYD + NBD, based on the published Nlrp3 structure (Sharif et al.,
2019). was obtained from the SWISS-MODEL website, a homology-modeling server
(https://swissmodel.expasy.org/) (Waterhouse et al., 2018) The structure of Nlrp12’s PYD +
NBD was generated based on the homology between Nlrp12 and Nlrp3 , using amino acids 124 -
526. A peptide with Hck’s sequence Gln507-Serine (Ser)-Val-Leu-Asp511 was chosen as the
docking structure. These two molecules were prepared as docking molecules in
“Tools/Surface/Binding analysis/Doc prep” options in the University of San Francesco (UCSF)
Chimera program (version 1.13.1), with Nlrp12’s PYD + NBD as a “receptor” and Hck’s 5
amino acid peptide Gln507-Ser-Val-Leu-Asp511 as a “ligand”. Finally, the docking was
performed in Tools/Surface/Binding analysis/AntoDock Vina” options in UCSF Chimera. The
structure of Hck’s 5 amino acids Gln507-Ser-Val-Leu-Asp511 was non-fixed, meaning that the
structure maintained its structure as it was found in full-length Hck.

Cell transfection
293T cells, THP-1 cells, U937 cells, and RAW 264.7 cells were seeded at a density of 5 x 10
5

per well in a 6-well plate. Transfection followed the Lipofectamine® 3000 reagent protocol with
the following modifications. Briefly, 3.75 µl of Lipofectamine® 3000 was added into 125 µl of
Opti-MEM® medium (Thermo Fisher Scientific). 2.5 µg of total plasmid DNA (prepared by
isolation through cesium chloride (CsCl) density gradient purification (Brakke, 1951)) (1.25 µg
of each plasmid, if there were two plasmids) and 5 µl of P3000
TM
reagent were added into
another aliquot of 125 µl of Opti-MEM® medium. Then these two tubes were mixed together,
37
and the mixed tube was incubated at room temperature for 15 min. 250 µl of this mixture was
added to the cells. The cells were incubated at 37 °C with 5% CO2 for two days until analysis.

For the luciferase assay, 293T cells were seeded at a density of 3 x 10
5
per well in a 24-well
plate. A 0.75 µl of Lipofectamine® 3000 was added into 25 µl of Opti-MEM® medium (Thermo
Fisher Scientific). 500 µg of total plasmid DNA and 1 µl of Lipofectamine® 3000
TM
reagent
were added into another aliquot of 25 µl of Opti-MEM® medium.

In Figures 16, 17, 18, 19, 20, 293T cells were seeded at a density of 3 x 10
6
per well in a 10-cm
tissue culture dishes. Similarly, 22.5 µl of Lipofectamine® 3000 was added into 750 µl of Opti-
MEM® medium (Thermo Fisher Scientific). 2.5-10 µg of total plasmid DNA, depending on the
experimental conditons, and 30 µl of P3000
TM
reagent were added into another aliquot of 750 µl
of Opti-MEM® medium.

Nlrp12 stable cell lines
The plasmid pEFIRES-P-puro-Nlrp12-3FLAG was made into a lentivirus by transfecting
pEFIRES-P-puro-Nlrp12-3FLAG alone with pVSV, the envelope plasmid, and pGAG and
pREV, the packaging plasmids, into 293T cells (see above for transfection method). The
lentivirus was then used to transduce the pEFIRES-P-puro-Nlrp12-3FLAG plasmid into THP-1
cells or U937 cells. pEFIRES-P-puro-Nlrp12-3FLAG was directly transfected into the RAW
264.7 cells. 2.5 µg/ml puromycin (Invitrogen
TM
by Thermo Fisher Scientific) was added.
pEFIRES-P-hygro-Hck was made into a lentivirus by transfecting pEFIRES-P-hygro-Hck alone
with pVSV, the envelope plasmid, and pGAG and pREV, the packaging plasmids, into 293T
38
cells (see above for transfection method). Subsequently, the lentivirus was used to transduce the
pEFIRES-P-hygro-Hck plasmid into THP-1 cells or U937 cells. pEFIRES-P-hygro-Hck was
directly transfected into RAW 264.7 cells, and 200 µg/ml hygromycin (Gibco
TM
by Thermo
Fisher Scientific) was added.

pEFIRES-P-puro-Nlrp12-3FLAG + pEFIRES-P-hygro-Hck plasmids were made into a lentivirus
by transfecting pEFIRES-P- puro-Nlrp12-3FLAG + pEFIRES-P-hygro-Hck plasmids with
pVSV, the envelope plasmid, and pGAG and pREV, the packaging plasmids, into 293T cells
(see above for transfection method). Subsequently, the lentivirus was used to transduce
pEFIRES-P-puro-Nlrp12-3FLAG + pEFIRES-P-hygro-Hck plasmids into THP-1 cells or U937
cells. pEFIRES-P- puro-Nlrp12-3FLAG + pEFIRES-P-hygro-Hck were directly transfected into
RAW 264.7 cells, and 2.5 µg/ml puromycin and 200 µg/ml hygromycin were added. After two
weeks, the cells were considered to be stably expressing Nlrp12, Hck, or Nlrp12 + Hck after
screening for expression of the proteins by western blot. The pEFIRES-P-hygro-Hck stable cells
were maintained by adding 200 µg/ml hygromycin. The pEFIRES-P-puro-Nlrp12-3FLAG and
pEFIRES-P-hygro-Hck stable cells were maintained by adding 2.5 µg/ml puromycin and 200
µg/ml hygromycin.

Vector only cell lines, in which pEFIRES-P-puro, pEFIRES-P-hygro, or pEFIRES-P-puro +
pEFIRES-P-hygro were stably expressed, was generated in the same way as above. The
pEFIRES-P-puro-Nlrp12-3FLAG stable cells were maintained by adding 2.5 µg/ml puromycin.
The vector only cells were maintained by adding 2.5 µg/ml puromycin, 200 µg/ml hygromycin,
or 2.5 µg/ml puromycin + 200 µg/ml hygromycin. All the stable cells were “pooled” clones.
39
Knock-down cells
Delivery of Hck siRNA and control random siRNA to U937 cells were performed by Neon
TM

transfection system (ThermoFisher). The protocol can be found online
(https://assets.thermofisher.com/TFS-Assets/LSG/manuals/neon_device_qrc.pdf). The siRNAs
and the controls were from Jae Jung’s lab.

Cell lysis and immunoprecipitation
After trypsinization, cells were lysed in 500 µl of lysis buffer (1% NP-40, 4mM Tris-HCl, pH
8.0, 150 mM NaCl). Cells were then treated with freeze-thaw cycles, i.e., frozen in the -80°C
freezer for 5 min and thawed at 37°C in a water bath for 3 min, for a total of five times. Cells
were then centrifuged at 12,000 x g for 10 min at 4°C. After centrifugation, the supernatant was
saved at -20°C

until further use.

For immunoprecipitations, cell lysates were pre-cleared by adding 40 µl of protein A/G agarose
beads (Thermo Fisher Scientific) from a 50% slurry, and the sample was rotated for 1 hr at 4°C.
The sample was spun at 1000 x g for 1min. Then, 30 µl of pre-cleared whole cell lysate (WCL)
were removed and run on immunoblotting as a control. The remaining cell lysate was incubated
with 1 µg anti-Hck mouse monoclonal primary antibody overnight. On the second day, 30 µl of
protein A/G agarose, from a 50% slurry and previously washed with lysis buffer three times,
were added to the tube, and the tube was incubated for 3 hrs at 4°C. The beads were then washed
with lysis buffer, but with the NaCl concentration increased to 500 mM. The tube was briefly
centrifuged at 1000 x g for 1min., and the supernatant was removed. This step was performed for
40
a total of 5 times. The proteins were eluted from the beads by addition of SDS-PAGE sample
buffer.

Immunoblotting
Protein concentrations were determined by the Pierce
TM
BCA protein assay (Thermo Fisher
Scientific). 20µg of cell lysate samples and the entire immunoprecipitated samples were loaded
onto the 8% acrylamide gel. After overnight transfer of the proteins from an SDS-gel to a 0.45
µm pore size polyvinylidene difluoride (PVDF) membrane, the membrane was blocked for 1 hr
at room temperature with 5% non-fat dry milk dissolved in Tris-buffered saline with Tween 20,
pH=8.0 (TBST) (Sigma-Aldrich). Mouse monoclonal anti-V5 antibody (diluted 1:1000), the
rabbit monoclonal anti-V5 antibody (diluted 1:1000), mouse monoclonal anti-FLAG antibody
(diluted 1:1000), mouse monoclonal anti-Hck antibody (diluted 1:1000), mouse monoclonal anti-
Csk antibody (diluted 1:1000), the mouse monoclonal anti-4G10 antibody (diluted 1:1000), the
mouse monoclonal anti-p-Tyr-100 antibody (diluted 1:1000), the rabbit polyclonal anti-p-Y416
Src (diluted 1:1000), the rabbit polyclonal anti-p-Tyr521 Src antibody (diluted 1:1000), the
mouse monoclonal anti-phospho-IκB-α antibody (diluted 1:1000), the rabbit polyclonal anti-IκB-
α antibody (diluted 1:1000), the mouse monoclonal anti-GAPDH antibody (diluted 1:1000), or
the mouse monoclonal anti-β actin antibody (diluted 1:1000) were added individual,y and the
membranes incubated overnight. For detection of bands in the cell lysate, rabbit anti-
mouse IgG HRP-linked secondary antibody (diluted 1:2000), goat anti-rabbit IgG HRP-linked
secondary antibody (diluted 1:3000), or mouse IgGκ binding protein-HRP secondary antibody
(only for anti-Hck antibody) (diluted 1:1000) were added, and the membrane was incubated for
1hr at room temperature. For immunoprecipitations, Clean-Blot™ IP detection reagent (HRP)
41
secondary antibody (1:300) (Thermo Fisher Scientific), rabbit anti-mouse IgG HRP-linked
secondary antibody (diluted 1:2000), or goat anti-rabbit IgG HRP-linked secondary
antibody (diluted 1:3000) were used. The detection signal was visualized by using enhanced
chemiluminescence (ECL) reagent (Thomas Scientific, Swedesboro, NJ). Immunoblots were
analyzed by chemiluminescence on a Bio-Rad ChemiDoc
TM
Touch Gel imaging system (Bio-
Rad, Hercules, CA).

Immunofluorescence
A protocol for immunofluorescence was followed
(https://www.cellsignal.com/contents/resources-protocols/immunofluorescence-general-
protocol/if), with the following modifications. Coverslips were autoclaved and coated with 0.1
mg/ml of poly-D-lysine hydrobromide (mol wt 70,000-150,000) (Sigma-Aldrich). Cells were
plated onto the coverslips. Fixation was stopped by adding 50 mM NH4Cl in PBS for 15 min at
room temperature. Fixed cells were permeabilized by adding 0.5% Triton®-X-100 in PBS for 10
min. The cells were then washed with PBS three times and blocked with 0.1% of BSA in PBS at
room temperature for 1 hr. The rabbit monoclonal anti-FLAG antibody (diluted 1:100) and
mouse monoclonal anti-Hck antibody (diluted 1:100) were added to each coverslip and incubated
overnight. Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, conjugated to Alexa
Fluor 488 (diluted 1:100) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody,
conjugated to Alexa Fluor 555 (1: 100) were added. After incubation with secondary antibodies,
the cells were washed five times with PBS. During the first step, DAPI (Invitrogen
TM
by Thermo
Fisher Scientific) was also added at 1:1000 in PBS for 2 min. Finally, the coverslips were
mounted in ProLong™ Gold Antifade mounting medium (Life Technologies Corporation by
42
Thermo Fisher Scientific, Eugene, OR). The stained slides were viewed on a ZEISS LSM 880
(Pleasanton, CA) with Airyscan. The images were viewed and processed using Fiji (Fiji is just
ImageJ) software (Schindelin et al., 2012).

Ingenuity Pathway Analysis (IPA) diagram
Yeast two-hybrid data were analyzed by IPA software (QIAGEN Inc.,
https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

Bioinformatic analysis
All of the clinical samples were download from the cBioPortal website (Cerami et al., 2012b;
Gao et al., 2013; The Cancer Genome Atlas Research Network, 2013)
(https://www.cbioportal.org) and Oncomine website (https://www.oncomine.org) (Rhodes et al.,
2004). The data were then analyzed through Prism 6.0 (San Diego, CA, USA).

Luciferase assay
One day after transfection, the Dual-Glo luciferase assay was performed (Promega, USA).
Before the experiment, the Dual-Glo luciferase buffer was added to defrosted Dual-Glo
luciferase substrate (“Dual-Glo” reagent). Then “x” µl of “Dual-Glo Stop and Glo” substrate was
added into 100x µl “Dual-Glo Stop and Glo” buffer (“Dual-Glo Stop and Glo” reagent). “X”
depended on how much Dual-Glo reagent was needed on the experimental day. During the
experiment, a 96-well plate of cells was taken out form 37°C incubator and was equilibrated to
room temperature. Then 75 µl of Dual-Glo reagent was added into each well of the plates. After
waiting for 15 mins, firefly luminescence was measured using a Synergy H1 Hybrid Multi-mode
43
Reader Machine (BioTek® Instruments Inc, USA). And then 75 µl of “Dual-Glo Stop and Glo”
reagent was added into the each well of the 96 wells. After waiting for another 15 mins, Renilla
luminescence was measured using the same machine.

Real-time (RT)-PCR
RNA was extracted using TRIzol reagent (Ambion® by Life Technologies). Briefly, the cells
that were collected from 6 well plates (about 1-2 million) was put in 1000 µl of TRIzol reagent
and incubated for 5 min in a microcentrifuge tube. Then 200 µl of chloroform (Mallinckrodt
ChronAR® Inc, USA) was added, and the tube was shaken for 15 seconds. After waiting for 15
mins at room temperature, the tube was centrifuged for 15 min at 12,000 x g at 4°C. After
centrifugation, the tube was divided by three layers: the top clear layer; the middle white layer;
and the low red layer. The top layer (about 500 µl) was removed to another microcentrifuge tube,
to which was added 500 µl of isopropanol (Sigma-Aldrich, USA). The tube was vortexed and
then waited for 10 mins at room temperature. Then the tube was centrifugated at 12,000 xg for
10 min at 4°C. The pellet was washed with 1ml of 70% ethanol and centrifuged at 12,000 xg for
5 min at 4°C. The pellet was dried for 10 min. Finally, about 20 µl distilled water was added into
pellet.

For cDNA synthesis: 1 ug RNA and 1 µl of 50 µM hexamer (Thermo Fisher Scientific) were
mixed together and incubated at 70 °C for 5 min in a ProFlex PCR System (Applied Biosystems
by Life Technologies). As the PCR machine cooled to 4°C, the tube was taken out and in a new
tube, the following reaction was set up: the previously mixed 1ug RNA and 1 µl hexamer, 2 µl of
5x Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptase buffer (Promega), 0.5 µl of
44
M-MLV reverse transcriptase (Promega), 1µl of 10 mM each of dNTP (Thermo Fisher
Scientific), 1 µl of random primers (Invitrogen by Thermo Fisher Scientific), 1 µL of RNase
inhibitor (Applied Biosystems by lLife Technologies), and UltraPure™ DNase/RNase-free
distilled water to bring the total reaction volume to 10 µl. The reaction was returned to the PCR
machine, run at 37 °C for 1 hr, and then kept at 4°C indefinitely.

For the RT-PCR reaction, 1µl of cDNA was used, and added to 1µl of 10 µM of forward/reverse
primers mix, 5 µl of iQ SYBR green supermix 2X (Biorad), and UltraPure™ DNase/RNase-free
distilled water to bring up the total volume to 10 µl. The tube was put in the CFX96TM real-time
PCR detection system and the procedure was set up following the iQ SYBR green supermix
Users Guide with modifications, with the following reaction conditions: 95°C for 3 min, 40
cycles of 95°C for 10 s, 55°C for 45 s, and 72°C for 10 min for extension.
The sequences of the forward/reverse primers were as follows:
IL-6 forward: ATGAACTC CTTCTCCACAAGC
IL-6-reverse: GTTTTCTGCCAGTGC CTCTTTG (from (Safley et al., 2004))
IL-1β forward: ACAGATGAAGTGCTCCTTCCA
IL-1β reverse: GTCGGAGATTCGTAGCTGGAT (from (Li et al., 2004)
TNFα forward: CTTCTGCCTGCTG CACTTTGGA
TNFα reverse: TCCCAAAGTAGACCTGCCCAGA
β-actin forward: TGAC GGGGTCACCCACACTGTGCCCATCTA
β-actin reverse: CTAGAAGCATTTGC GGTGGACGATGGAGGG (from (Cowan et al., 1997)

45
AlamarBlue® assay
AlmarBlue® assay is a cell viability assay (Thermo Fisher Scientific) and was performed
according to the manual’s instruction. Briefly, 90 µl of 1 x 10
4
cells was seeded into the one well
of a 96-well plate. And then 10 µl of AlamarBlue® reagent was added into each well. The plate
was incubated at 37°C for 2 hrs, protecting it from light. And then the fluorescence excitation
was read at 540 nm and 570 nm, and fluorescence emission was read at 580 nm and 610 nm.
Each experiment was done at least three times.

Cell counting using a hemocytometer
The hemocytometer was purchased from Warner-Lambert Technologies Inc. (Buffalo, NY). The
cells were first diluted in PBS 10 times. And then 15 µl of cell medium plus 15 µl of 0.4% of
trypan blue (Thermo Fisher Scientific) were mixed in a microcentrifuge tube. After 10 µl of the
mixture were applied to the hemocytometer, the number of live, unstained cells were counted at
one square of a total of 9 squares. The counting was repeated four times. The cell number was
calculated as:
The number of the undiluted cells = average number of live, unstained cells from four times of
counting x 10
4
x 2 x 10.

Mice work and blood collection
Work on mice was approved by the University of Southern California Institutional Animal Care
and Use Committee (IACUC). Mice of WT (C57BL/6) and Nlrp12 -/- strains were transferred
from a post-doctoral fellow in Dr. Jae Jung’s lab. 200 µl of blood was obtained from wild type
and Nlrp12-/- littermates, by orbital puncture using a capillary (Thermo Fisher Scientific). The
46
collected blood with 20 µl of 0.5M ethylenediaminetetraacetic acid (EDTA) was put in the 200
µl of blood in 1.5 microcentrifuge tubes.

Flowcytometry
The blood sample was briefly centrifuged, and the drops on the wall of the tube were also
collected. 150 µl was transferred to a 5 ml centrifuge tube. 4 ml of 1x red blood cell (RBC) lysis
buffer that was freshly diluted from 10x RBC-lysis buffer was added (Affymetrix, San Diego).
The tube was vortexed and incubated at room temperature for 10 min. While waiting for the
incubation, 1.2 µl of primary antibodies were mixed in 200 µl of flow cytometry buffer (4% fetal
bovine serum (FBS) in Dulbecco's phosphate-buffered saline (DPBS)) in a 1.5 microcentrifuge
tube (Table 2). The antibodies mixture in the flow cytometry buffer was prepared under
protection from light, and vortexed. 3.0 µl of antibodies are for one sample of each genotype.
The cells were centrifuged at 652 x g for 5 min at room temperature. The supernatant was
discarded, and 200 µl of antibody mix was added into each set.

For the compensation group (the tube only contained antibodies and fluorescence-activated cell
sorting (FACS) buffer), the tube was incubated for 30 min on ice covered with foil to protect the
antibodies from light. After the incubation, 4 ml of FACS buffer was added into the tube. The
tube was centrifuged at 652 x g for 5 min at room temperature, and the supernatant was
discarded. 200 µl of FACS buffer was added to the cells, and then the cells were ready for
analysis in a Becton-Dickinson (BD) LSR II flow cytometry machine (BD Biosciences, San
Jose, CA) for FACS.

47

Table 2: Antibodies added into the flow cytometry buffer to detect blood leukocyte
composition
Antibody colors
Antibodies (set 1 for detecting neutrophils, monocytes, and
dendritic cells)
Fluorescein
isothiocyanate
(FITC)
CD19, CD3
Allophycocyanine (APC) Lymphocyte antigen 6 complex locus G6D (Ly-6G)
APC-Cyanine (Cy) 7 CD45
eFluor 450 CD11b

48
Statistical analysis
The Spearman coefficient and Pearson coefficient used for co-expression was generated by
cBioPortal website(2013; Cerami et al., 2012b; Gao et al., 2013). Clinical samples from the
Oncomine website (https://www.oncomine.org) were downloaded and checked for Nlrp12
expression levels and Hck levels. The Fisher exact test and the odds ratio test used for co-
occurrence were generated by the cBioPortal website. Analysis of variance (ANOVA) was
performed to check whether a significant difference of the treatment exists among all the groups.
A p-value < 0.05 was considered statistically significant. * indicated p-value < 0.05. ** indicated
p-value < 0.01. *** indicated p < value < 0.001. If P < 0.05, then the Tukey test was used to
determine whether a pair-wise difference exists. Quantification of the western blot bands was
performed by FIJI software (Schindelin et al., 2012) and made into graphs by Prism 6.0
(GraphPad Software, Inc., San Diego, CA).

49
Chapter 4: Nucleotide binding domain and leucine-rich repeat pyrin domain-containing
protein 12: characterization of its binding to hematopoietic cell kinase

Introduction
PRR are the first major line of cellular defense against DAMP and PAMP (Takeuchi and Akira,
2010). Among PRR, one class of proteins is the NBD and LRR receptor family, Nlrp12 is one
protein that belongs to the PYD subfamily of the NLR class and is a multi-functional protein
(Tuncer et al., 2014). Nlrp12’s functions likely all depend on defined protein-protein
interactions. For example, in anti-inflammatory functions of Nlrp12, Nlrp12 interacts with
TRAF3 and NIK in the non-canonical NF-κB pathway, when TLR are stimulated with
Pam3Cys4 triacylated lipopeptides followed by addition of CD40 (Allen et al., 2012). Nlrp12’s
inhibition of the non-canonical NF-κB pathway, thereby effecting an anti-inflammatory function,
has been reported in many publications (Allen et al., 2012; Arthur et al., 2010; Chen et al., 2017;
Chen et al., 2019; Jeru et al., 2011; Truax et al., 2018; Vladimer et al., 2012; Ye et al., 2008;
Zaki et al., 2011). For example, in colitis-driven colon cancer, Nlrp12 -/- mice experience a
higher frequency of death, weigh less, have a shorter colon length, and have more severe disease
progression than WT mice, all of which may occur through Nlrp12’s inhibition of the non-
canonical NF-κB pathway (Allen et al., 2012; Zaki et al., 2011). In addition, Nlrp12 -/- mice
have been reported to exhibit higher innate immunity when it is infected by the virus because
Nlrp12 inhibits the non-canonical NF-κB pathway; thus, in a Nlrp12 -/- mice, a more significant
increase of NF-κB activity is observed compared to WT mice when it is infected by the virus
(Chen et al., 2019). And in the case of Nlrp12 protecting the diversity of intestinal microbiota
(Chen et al., 2017), thereby preventing obesity (Truax et al., 2018), it has also been reported that
50
Nlrp12 -/- mice display significantly decreased intestinal microbial diversity due to increased
innate immunity, resulting from the lack of inhibition of the non-canonical NF-κB pathway.

In addition, Nlrp12 also interacts with IRAK1 in the canonical NF-κB pathway when TLRs are
stimulated, also to inhibit the inflammatory response (Ye et al., 2008). In contrast, for human
studies of Nlrp12-associated autoinflammatory disorders, no protein-protein interactions have
been reported (Jeru et al., 2008; Jeru et al., 2011). Functionally, however, Nlrp12 -/- mice had a
higher immunological response in the autoimmune model of contact hypersensitivity (Arthur et
al., 2010); thus, these mice are more prone to develop contact hypersensitivity. This results most
likely from Nlrp12 -/- mice having defective migration of their DCs. But, in the mouse study, no
protein-protein interactions had been reported either (Arthur et al., 2010). And Nlrp12 is
protective against Yersinia pestis infection through Nlrp12’s activation of the inflammasome
which results in secretion of IL-18 and IL-1β. But again, no protein-protein interactions of
Nlrp12 within inflammasomes had been characterized (Vladimer et al., 2012). Thus, there is a
further need to find and to characterize previously unreported Nlrp12-binding proteins.
Knowing Nlrp12’s protein binding partners may help to gain mechanistic insight into pathways
previously characterized as being regulated by Nlrp12. Moreover, identifying and validating
potentially novel binding partners of Nlrp12 may suggest novel functions for Nlrp12.

In humans, Nlrp12 is mainly expressed in macrophages, DCs, and neutrophils. In mice, Nlrp12 is
primarily expressed in DCs and neutrophils (Arthur et al., 2010; Chen et al., 2019; Hornick et al.,
2018). The Nlrp12 protein has three domains, from N- to C- terminus: Pyrin domain (PYD),
NBD, and LRR domains (Damiano et al., 2004; Janowski et al., 2013; Tuncer et al., 2014). It is
51
generally accepted that the LRR domain, like Nlrp1’s LRR domain and Nlrp3’s LRR domain, is
responsible for sensing ligands or other types of activators of Nlrps in modulating their role in an
immunologic response (Faustin et al., 2007; Kanneganti et al., 2007; Martinon et al., 2004;
Sharif et al., 2019). However, interestingly, it has been reported that without the LRR domain,
Nlrp3 still can be activated by triggers of the canonical NF-κB pathway (Hafner-Bratkovič et al.,
2018). The NBD domain is responsible for self-oligomerization when Nlrp is activated, and the
PYD domain is responsible for regulating downstream signaling pathways (and thus, it is called
the “interaction domain”) (Hafner-Bratkovič et al., 2018; Hornick et al., 2018). Here we used a
truncated form of Nlrp12 (PYD + NBD) to identify possible protein-protein interactions that may
regulate signaling pathways in which Nlrp12 is involved. In addition, to our knowledge, there are
currently no protein-protein interaction studies that have been performed for this truncated form
of Nlrp12 and may thus represent a new general approach to screening for protein-protein
interactions involved in signaling complexes with Nlrps.

A yeast two-hybrid screen was performed to identify potential binding partners of Nlrp12’s PYD
+ NBD. Hck, a member of the Src family of non-receptor tyrosine kinases, was one of the top
positive clones (“hits”) among all of the proteins that appeared to interact with Nlrp12. Further
experiments were done to confirm the novel binding of Nlrp12 to Hck and to characterize key
structural features of the binding interaction, including simulation of a 5 amino acid fragment of
Hck Gln507-Ser-Val-Leu-Asp511 docking to Nlrp12’s PYD + NBD domains. In consideration
with a bioinformatics analysis included here, a potential interaction between Nlrp12 and Hck
may be involved in modulating the role of Hck in the pathogenesis of AML (Lopez et al., 2016;
Roversi et al., 2015; Roversi et al., 2017).
52
Results
Nlrp12 interacts with Hck in the yeast two-hybrid assay
A human leukocyte cDNA library was used as prey in the screening of binding partners for
Nlrp12 using the yeast two-hybrid assay. The basic principle of the yeast two-hybrid assay is that
if the cDNA library contains clones that express proteins, or parts thereof, that interact with the
co-expressed bait, the Nlrp12 PYD + NBD (Figure 5A) in this case, then the co-transformed
yeast clones will grow. The cDNA clones from the library that supported growth would then be
isolated and characterize, to identify what proteins interact with Nlrp12’s PYD + NBD. The
leukocyte library was used because Nlrp12 is mainly expressed in leukocytes in humans
(Hornick et al., 2018; Linz et al., 2017; Normand et al., 2018; Tuncer et al., 2014), and thus
putative binding partners for Nlrp12 are also likely to be expressed there.

The screening resulted in 119 positive clones from the leukocyte cDNA library (“hits”)
expressed from 48 distinct genes (Table 3) that were subsequently identified as encoding for
proteins (or portions thereof) that potentially interact with Nlrp12’s PYD + NBD. The clones
with the most hits are listed in Table 4. Among the 119 hits, which included repeated hits of the
same genes, 9 were individual clones that encoded fragments of a member of the Src family of
non-receptor tyrosine kinases, Hck, which was among the highest number of hits for a single
gene (Figure 5B and Table 4). Moreover, of the 9 positive Hck clones, 5 were distinct Hck
fragments (Figure 5C). Full-length Hck p61 was also obtained as a hit in another independent
yeast two-hybrid screen (data not shown). Hck has two isoforms, p59 and P61, in Homo sapiens.
P61 and p59 share the same transcript, but p61 uses an uncommon start codon CTG, and p59
uses the common one, ATG (Robbins et al., 1995), to give rise to two distinct transcripts.
53
Endogenously expressed in phagocytes, Hck p59 is mainly localized to the plasma membrane,
while Hck p61 is primarily localized to lysosomes, and both of them are also found in the Golgi
apparatus (Carreno et al., 2000). It has been suggested that overexpression of p59 promotes the
formation of membrane protrusions such as pseudopodia and overexpression of p61 induces
podosome rosettes and triggers actin polymerization around lysosomes (Carreno et al., 2000;
Guiet et al., 2008). However, there are no differences between p61 and p59 in the region of Hck
that appears to bind to Nlrp12’s PYD + NBD (see below), the C-terminal part of Hck. Thus,
detecting a full-length clone of only p61 as a hit, opposed to p59, appeared to be coincidental.

54

Figure 5: Yeast two-hybrid prey, bait, and results. (A). Schematic drawing of the domain
structure of full-length Nlrp12 (top) and the domains used as the construct for the yeast two-
hybrid screen (bottom). (B).The domain structures of the two alternatively spliced isoforms of
Hck, Hck p59 and Hck p61. (C). Identification of the clones of Hck interacting with Nlrp12
(“hits”) obtained from the yeast two-hybrid screen. Among the nine of the clones positive for
Hck, one clone encoded for the smallest interacting fragment of Hck, the C-terminal 40 amino
acids of Hck. The numbers in parentheses indicate the total number of hits obtained in the screen.

55
Table 3: Complete yeast two hybrid results
Name Frequency
Deleted in azoospermia associated protein 2 (DAZAP2) 10
Filamin A, alpha (FLNA) 9
HCK 9
Chaperone DnaJ (a.k.a., HSP40) homolog, subfamily B, member 1 (DNAJB1) 9
Metallothionein 2A (MT2A) 7
CD14 6
DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5) 6
Talin 1 (TLN1) 5
Calcium modulating ligand (CAMLG) 4
Squamous cell carcinoma antigen recognized by T cells (SART1) 4
Ras-related nuclear protein binding protein 9 (RANBP9) 3
TRAF3IP3 3
Thyroid hormone receptor interactor 4 (TRIP4) 3
Ribosomal protein S20 (RPS20) 2
Coronin, actin binding protein, 1A (CORO1A) 2
Glutamate-ammonia ligase (GLUL) 2
Actin, beta (ACTB) 2
WW domain containing adaptor with coiled-coil (WAC) 2
Drosha, ribonuclease type III (DROSHA) 2
56
DnaJ (HSP40) homolog, subfamily A, member 3 (DNAJA3) 1
PTP receptor type E 1
Ubiquilin 1 (UBQLN1) 1
Adenylate cyclase-associated protein 1 (CAP1) 1
A kinase (protein kinase A (PRKA)) interacting protein 1 (AKIP1) 1
Ubiquilin 2 (UBQLN2) 1
Baculoviral inhibitor of apoptosis (IAP) repeat containing 3 (BIRC3) 1
Trinucleotide repeat containing 6C (TNRC6C) 1
Alkaline phosphatase, liver/bone/kidney (ALPL) 1
Pre-mRNA processing factor 6 (PRPF6) 1
Ubiquitin-like (UBX) domain protein 2B (UBXN2B) 1
B-cell lymphoma (BCL) 2-associated athanogene 6 (BAG6) 1
Secreted and transmembrane 1 (SECTM1) 1
Spermidine/spermine N1-acetyltransferase family member 2 (SAT2) 1
Zinc finger protein 426 (ZNF426) 1
Major histocompatibility complex, class I, B (HLA-B) 1
Fibrillin 2 (FBN2) 1
SH3-domain binding protein 2 (SH3BP2) 1
Myosin IF (MYO1F) 1
Ferritin, heavy polypeptide 1 (FTH1) 1
Leucine rich repeat containing 42 (LRRC42) 1
Fermitin family member 3 (FERMT3) 1
57
Lectin, galactoside-binding, soluble, 9B (LGALS9B) 1
Superoxide dismutase 2, mitochondrial (SOD2) 1
Carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15 (CHST15) 1
Hematopoietic cell-specific Lyn substrate (HCLS) 1 associated protein X-1 (HAX1) 1
Major histocompatibility complex, class I, C (HLA-C) 1
Splicing factor, arginine/serine-rich 17A (SFRS17A) (a kinase anchoring protein 17A) 1
IL-8 1

58

Table 4: Yeast two-hybrid results (hit frequency >=3)
Name Frequency
DAZAP2 10
FLNA 9
HCK 9
DNAJB1 9
MT2A 7
CD14 6
DDX5 6
TLN1 5
CAMLG 4
SART1 4
RANBP9 3
TRAF3IP3 3
TRIP4 3

59
In directed screens using the yeast two-hybrid assay, Nlrp12’s PYD + NBD domain binding to
Hck C-terminal 40 amino acids appears to be selective for Nlrp12 and Hck binding to Nlrp12
appears to be selective for Hck
Initially, Hck C1 fragment was the shortest Hck fragment from a positive clone that interacted
with the bait, Nlrp12’s PYD + NBD (Figure 6A). Nlrp 3 and Nlrp8 were chosen based on the
phylogenetic trees of the PYD + NBD domains of members of the Nlrp family: phylogenetically,
Nlrp3’s PYD + NBD is the most closely related Nlrp PYD + NBD to that of Nlrp12, while
Nlrp8’s PYD + NBD is among the furthest related PYD + NBD to that of Nlrp12 (Figure 6B). In
Figure 6C, yeast clones grew on the 4 drop out (DO) and 4DO + 3-AT high stringency plates
when the yeast were co-transformed with Nlrp12’s PYD + NBD and Hck C1 fragment; and thus,
this result is indicative of an interaction between the two constructs. However, yeast clones were
not observed on 4DO and 4DO + 3-AT plates when Nlrp3’s PYD + NBD and Hck C1 fragment
were co-transformed, nor when Nlrp8’s PYD + NBD and Hck C1 fragment were co-transformed.
These results suggest that the Hck C1 fragment binds to Nlrp12’s PYD + NBD, but not to those
from either Nlrp3 or Nlrp8, results consistent with a selective interaction between Nlrp12’s PYD
+ NBD and the Hck C1 fragment.

Similarly, yeast clones again grew on the high stringency 4DO and 4DO + 3-AT plates when the
Hck C1 fragment was co-transformed with Nlrp12’s PYD + NBD. However, clones did not grow
on the 4DO and 4DO + 3-AT plates when the C1 fragment of other members of the Src family of
non-receptor tyrosine kinases were co-transformed with Nlrp12’s PYD + NBD (Figure 6D). This
experiment suggests that the interaction of Hck C1 fragment with Nlrp12 is selective for Hck;
60
the C1 fragment of other members of the Src family of non-receptor tyrosine kinases did not bind
to Nlrp12’s PYD + NBD (Figure 6D).

61

Figure 6: Specificity of binding of Nlrp12 with Hck. (A). Schematic drawing of Hck (p61
isoform, top) and, for comparison, Hck C1 fragment (lower) used for testing the specificity of
binding of Hck to other members of the Nlrp family of proteins. The numbers represent the
amino acid residues of the protein or protein fragment. (B). Phylogenetic tree showing the
relationships among the PYD + NBD region of all of the members of the human Nlrp family of
62
proteins. The phylogenetic tree was produced through Clustal Omega
(https://www.ebi.ac.uk/Tools/msa/clustalo/). For each Nlrp family member, the PYD + NBD of
the longest isoform was selected for generating the phylogenetic tree. Nlrp3 is phylogenetically
the closest Nlrp to Nlrp12, while Nlrp8 is phylogenetically among the most distant from Nlrp12.
Nlrp3 and Nlrp8 were highlighted in red color. (C). When Nlrp12’s PYD + NBD and Hck C1
fragment were co-transformed into yeast, they grew on the highest stringency plates (4DO +
3AT), consistent with an interaction between the two constructs. On the other hand, neither
Nlrp3’s PYD + NBD co-transformed with the C1 fragment nor Nlrp8’s PYD + NBD co-
transformed the C1 fragment grew on the highest stringency plates. These results are consistent
with a specific interaction between Nlrp12 and the Hck C1 fragment, but not with the other
Nlrps.(D). Yeast clones grew on the high stringency plates 4DO + 3AT when Hck’s C1 fragment
and Nlrp12’s PYD + NBD were co-transformed, but they did not grow on the high stringency
4DO + 3AT plates when the C1 fragments of other members of the Src family of non-receptor
tyrosine kinases were co-transformed with Nlrp12’s PYD + NBD. These results are again
consistent with a specific interaction between Nlrp12 and Hck.

63
In directed screens using the yeast two-hybrid assay, amino acids F503, Q507, L510, and
D511 of Hck are critical for the binding between Nlrp12’s PYD + NBD and Hck C-terminal
40 amino acids
The Hck C1 fragment were divided into four helical regions according to its atomic structure
(Sicheri et al., 1997) (PDB accession number: 1AD5): R1, R2, R3, and R4 (designated by us.
The four regions in the Hck C1 fragment) (Figure 7A). As shown in the sequence alignments in
Figure 7B, the primary amino acid sequences of R2, R3, and R4 are very well conserved among
members of the Src family of non-receptor tyrosine kinases, while R1 is not. To characterize
further the important structural features of Hck’s binding to Nlrp12’s PYD + NBD, all of the
amino acids comprising each of the R2, R3, and R4 domains were mutated to alanine, but
mutated one section at a time, and were denoted as the Hck M2 fragment, Hck M3 fragment, and
Hck M4 fragment, respectively. The amino acids comprising the R1 region were mutated to
those amino acids in the R1 region of the Src family non-receptor tyrosine kinase Lyn, which has
the closest phylogenetic relationship to Hck (Figure 7C). All of the mutated constructs are
illustrated in Figure 7D. Yeast still grew on the high stringency 4DO + 3AT plates when
Nlrp12’s PYD + NBD was co-transformed with the Hck M1 fragment, Hck M2 fragment, and
Hck M4 fragment. But, yeast could not grow when Nlrp12’s PYD + NBD was co-transformed
with Hck M3 fragment (Figure 7E). These experiments suggested that the R1, R2, and R4
regions are dispensable for binding, while R3 may be important for binding. Further analysis was
conducted to identify individual critical amino acids of Hck’s R3 region binding to Nlrp12’s
PYD + NBD using alanine scanning mutagenesis. However, the intervening sequences between
R1, R2, R3, and R4 were not examined for binding.

64
To identify the key amino acid residues involved in binding of R3 to Nlrp12’s PYD + NBD,
each amino acid in R3 was individually mutated to alanine in an alanine (Ala) scanning
mutagenesis analysis, on the background of the Hck C1 fragment. As shown by the yeast two-
hybrid assay, the mutations of Phe503Ala, Gln507Ala, Leu510Ala, and Asp511Ala abolished the
growth of yeast clones on the high stringency 4DO when these yeast strains were co-transformed
with Nlrp12’s PYD + NBD (Figure 7F). These experiments suggest that these four amino acids
are critical for binding. On the other hand, yeast clones still grew on the high stringency 4DO
plates when the Hck C1 fragments with the Tyr505Ala, Ser508Ala, or Val509Ala mutations
were co-transformed with Nlrp12’s PYD + NBD (Figure 7F). These experiments suggest that
Tyr505, Ser508, and Val509 are dispensable for binding of Hck C1 fragment to Nlrp12’s PYD +
NBD. Whether Glu504 and Isoleucine (Ile) 506 are critical for binding is unfortunately not clear
as the yeast did not grow on the control plates (SD/-Trp-Leu). Overall, however, these results
suggest that the amino acids Phe503, Gln507, Leu510, and Asp511 in Hck are critical for the
binding of Hck C1 fragment to Nlrp12’s PYD + NBD.

On the other hand, these four amino acid residues are all conserved in all members of the Src
family of non-receptor tyrosine kinases (Figure 7A), suggesting that, while they are important for
binding, they cannot account for the specificity or selectivity of binding between Nlrp12 and
Hck. In addition, these four critical residues, as shown in Figure 7C, appear to be in the same
plane along a helix, which may then form an interface that is important for binding to Nlrp12,
provided the that structure of C1 is maintained when the region is expressed alone. However, it is
unclear whether the structure of the isolated Hck C-terminus used in the screens here will retain
its structural features that it possesses in full-length Hck, due to the loss of intramolecular
65
interactions for the C-terminal region. Moreover, for example, the amino acids Arg500, Pro501,
Thr502, Asp512, Thr515, Gln520, and Tyr521, are 100% conserved among all other members of
the Src family of non-receptor tyrosine kinases and are therefore unlikely to play a role in
specificity and selectivity of binding of Nlrp12 with Hck. But, it is possible that two or more
amino acids in the intervening sections between the R1-R4 regions, which were not analyzed by
mutagenesis, may then actually account for the specificity and selectivity of binding of Hck to
Nlrp12. One example is that the combination of Arg496, Pro497, and Gln522 is unique to Hck
compared to other members of the Src family of non-receptor tyrosine kinases.

66

Figure 7: Four amino acids in Hck C terminals are critical for binding to Nlrp12’s PYD +
NBD domain. (A). Structure of the Hck C1 fragment generated by Chimera from the UCSF
67
(version 1.13.1), extracted from its structure within the entire Hck protein. Hck C1 fragment is
thus divided into four helical domains: R1, R2, R3, and R4. One letter abbreviation of amino
acids was used here for convenience. The side chains of amino acids Phe (F) 503, Gln (Q) 507,
Leu (L)510, and Asp (D) 511 were shown. However, it is not clear whether the C1 fragment
retains the structural features shown here when it expressed on its own. (B). Primary amino acid
sequence alignments showing of the C1 fragments of all members of the Src non-receptor
tyrosine kinase family (40 amino acids for Hck, Blk, Lck, and Lyn; 42 amino acids for Fgr, Src,
Yes, and Fyn). The black lines indicate the R1, R2, R3, or R4 regions. The arrows denote the
four critical amino acids for binding of Hck C1 fragment to Nlrp12’s PYD + NBD. (C).
Phylogenetic tree showing the relationship among all of the C1 fragments of the members of the
Src family of non-receptor tyrosine kinases. The phylogenetic tree was produced through Clustal
Omega (https://www.ebi.ac.uk/Tools/msa/ clustalo/). Hck was highlighted in red color. (D). Four
sets of distinct mutations were made within the Hck C1 fragment. M1: the amino acids in the R1
region were mutated to the amino acids identical to the R1 region in Lyn (highlighted in red
color); M2: the amino acids in the R2 region were all mutated to Ala; M3: the amino acids in the
R3 region were all mutated to Ala; and M4: the amino acids in the R4 region were all mutated to
Ala. (E). Yeast did not grow on high stringency 4DO + 3AT plates when Hck C1 fragment with
the M3 mutations was co-transformed with Nlrp12’s PYD + NBD. But yeast can grow on the
high stringency 4DO + 3AT plates when Hck C1 fragment contained either M1, M2, or M4
mutations, suggesting that the R3 region within the C1 fragment was necessary for binding to
Nlrp12 NBD + PYD. (E). Yeast did not grow on high stringency 4DO plates when Nlrp12’s
PYD + NBD was co-transformed with Hck C1 fragment with single Ala substitutions at either
F503, Q507, L510, or D511. But yeast grew on high stringency 4DO plates when Nlrp12’s PYD
68
+ NBD was co-transformed with Hck’s C1 fragment in which the following amino acids in the
R3 region were individually mutated to Ala: Tyr (Y) 505, Ser (S) 508, or Val (V) 509. Yeast did
not grow on either 2DO nor 4DO plates when Nlrp12’s PYD + NBD was co-transformed with
Hck C1 fragment in which either Glu (E) 504 or Ile (I) 506 was individually mutated to Ala,
making the results with these particular mutants inconclusive.

69
In directed screens using the yeast two-hybrid assay, the shortest fragment of Hck that binds
to Nlrp12’s PYD + NBD is its C-terminal 30 amino acids
The shortest form of Hck that bound to Nlrp12’s PYD + NBD was determined by observing
whether the yeast clones grew on high stringency 4DO or 4DO +3AT plates when Nlrp12’s PYD
+ NBD was co-transformed with Hck fragments C1 to C7 and N1 to N5 (Figure 8) (data of
Nlrp12’s PYD + NBD co-transformed with Hck fragment C7 is not shown). Hck’s C1 to C7
fragments were gradually truncated from the N-terminus of the Hck C-terminal 40 amino acid
fragment (Figure 8A and 8B). Hck N1 to N5 fragments were gradually truncated from the C-
terminus of the Hck C-terminal 40 amino acid fragment (Figure 8C and 8D). Yeast strains grew
on high stringency 4DO or 4DO +3AT plates when either the Hck C1, C2, or C3 fragment was
co-transformed with Nlrp12’s PYD + NBD. Thus, of all of the constructs screened, the shortest
fragment of Hck that can apparently bind to Nlrp12’s PYD + NBD is the C-terminal 30 amino
acids, as indicated by the Hck C3 fragment (Figure 8B and 8D). In addition, the C-terminal 5
amino acids also appear to be important for binding, as clones co-transformed with Nlrp12’s
PYD + NBD with N1, with a deletion of the C-terminal 5 amino acids of Hck, did not grow on
the high stringency 4DO and 4DO + 3AT plates (Figure 8D). The structures of both inactive Hck
(PDB accession number: 1AD5) and active c-Src (PDB accession number: 1Y57), the
prototypical member of this family of nonreceptor tyrosine kinases related to Hck, show that the
C-terminal 4 amino acids are surface-exposed, indicating that these four amino acids that are
critical for binding may available to interact with Nlrp12, regardless of the state of activation of
Hck.

70

Figure 8: The last 30 amino acids of Hck C terminal are critical for binding to Nlrp12’s
PYD + NBD domains. (A). Schematic drawing of Hck truncations comprising the C1 to C7
71
fragments. (B). Yeast grew on the high stringency 4DO plates and 4DO +3AT plates when Hck’s
C1 fragment, Hck’s C-terminal 35 amino acid fragment (C2), and Hck’s C-terminal 30 amino
acid fragment (C3), individually, were co-transformed with Nlrp12’s PYD + NBD. But yeast
did not grow on the high stringency 4DO plates nor 4DO+ 3AT plates when C4 to C7 fragments
were co-transformed to with Nlrp12’s PYD + NBD. The lack of yeast growth upon co-
transformation of the C7 fragment and Nlrp12 are not shown. (C). Schematic drawing of Hck
truncations comprising the N1 to N5 fragments. (D). Yeast grew on the high stringency 4DO
plates and 4DO +3AT plates when Hck’s C-terminal 40 amino acid fragment (C1) was co-
transformed with Nlrp12’s PYD + NBD (control). But yeast did not grow on the high stringency
4DO plates nor 4DO+ 3AT plates when N1 and N5 fragments were co-transformed to with
Nlrp12’s PYD + NBD, suggesting that the very C-terminal 5 amino acids are also necessary for
binding of Nlrp12. The colored boxes represent different fragments that were deleted each time.

72
Nlrp12 co-immunoprecipitates with Hck from mammalian cells, and Nlrp12 is co-localized
with Hck in mammalian cells
Nlrp12 and Hck could be co-immunoprecipitated when they were both heterologously co-
expressed in 293T cells (Figure 9A). Also, semi-endogenous conditions were used for co-
immunoprecipitations from cells in which Hck is endogenously expressed, and Nlrp12 was
heterologously stably expressed in epitope-tagged form, such as in macrophage-like RAW 264.7
cells (Figure 9B), monocyte-like THP-1 cells (Figure 9C), and lymphocyte -like U937 cells
(Figure 9D). Semi-endogenous conditions have been used in previous reports where Nlrp12 was
shown to interact with TRAF3 (Allen et al., 2012), NIK (Lich et al., 2007), and IRAK-1(Ye et
al., 2008). In all cases, Nlrp12 co-immunoprecipitated with the p59 and p61 isoforms of Hck, but
from these experiments, it is not clear that whether Nlrp12 interacts with p59, p61, or both
isoforms of Hck because the immunoprecipitating antibody was an anti-Hck antibody. In U937
cells and THP-1 cells, since heterologous Nlrp12 expression level was low, adding phorbol 12-
myristate 13-acetate (PMA) (1 µM for 24 hr) increased Nlrp12 expression level by stimulating
the PMA-sensitive cytomegalovirus promoter of Nlrp12’s expression plasmid (Figure 9C and
9D). Taken together, these data suggest that Nlrp12 and Hck (either p59, p61, or both isoforms)
can interact with each other in a mammalian cell system, a result that in part validates the
findings from the yeast two-hybrid assay.

After heterologous co-expression of Nlrp12 with either the p61 or the p59 isoform of Hck in
293T cells, immunofluorescent staining of Nlrp12 and Hck showed that Nlrp12 can be co-
localized with either p61 or p59 isoforms of Hck in most of the 293T cells that co-expressed both
proteins (Figure 9E and Figure 10). The co-localization of Nlrp12 and Hck is consistent with an
73
intracellular interaction between the two proteins. In addition, the lack of selectivity of Nlrp12
co-localizing with either p61 or p59 isoform of Hck is consistent with the results from the
screening assay, where the putative binding region of Hck to Nlrp12 is identical in both the p61
and p59 isoforms.
74

Figure 9: Nlrp12 co-immunoprecipitates with Hck and co-localizes with Hck by
immunofluorescence. (A). Nlrp12 co-immunoprecipitated with Hck when both proteins were
75
transiently exogenously co-expressed in 293T cells, with Nlrp12 having to be expressed as
epitope-tagged forms. Cells were lysed using a non-denaturing cell lysis buffer (see Material and
Methods). Co-immunoprecipitations were done using a mouse monoclonal anti-Hck antibody
followed by protein A/G agarose. The immunoprecipitated proteins were analyzed by
immunoblotting. The same method was used for experiments shown in (B), (C), and (D). (B).
Nlrp12 co-immunoprecipitated with endogenous Hck in macrophage-like RAW 264.7 cells,
when Nlrp12 is exogenously and stably expressed. Arrows show the two isoforms of Hck p59
and p61 that were immunoprecipitated by the anti-Hck antibody, although it is not clear whether
Nlrp12 co-immunopreciptitated with one form or the other, or both. (C). Exogenously and stably
expressed Nlrp12 co-immunoprecipitated with endogenously expressed Hck in monocyte-like
THP-1 cells. In these cells, PMA (1µM) was added to enhance the expression of Nlrp12 under
the PMA-sensitive cytomegalovirus promoter of the Nlrp12 plasmid. (D). Left panel shows that
Nlrp12 was exogenously and stably expressed in lymphoblast-like U937 cells. Right panel shows
that Nlrp12 co-immunoprecipitated with endogenously expressed Hck in U937 cells after PMA
was added to 1µM, as was done for THP-1 cells, to enhance expression of Nlrp12. (E).
Immunofluorescent images showing the colocalization of a cohort of Nlrp12 with either Hck p59
or p61. Hck was immunolabeled with a mouse monoclonal anti-Hck antibody, followed by a
secondary goat anti-mouse IgG (H+L) cross-adsorbed, conjugated to Alexa Fluor 555. For
Nlrp12, the primary antibody was a rabbit monoclonal anti-FLAG antibody, and the secondary
antibody was a goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, conjugated to
Alexa Fluor 488. Nuclei were counterstained by DAPI.

76

Figure 10: Immunofluorescent image of epitope-tagged Nlrp12-3FLAG and Hck co-
transfected into 293T cells, and fluorescently immunolabeled with anti-Hck and anti-FLAG
(Nlrp12) antibodies, and counterstained with DAPI. (A). Nlrp12 only transfected into 293T
77
cells. (B). Hck p61 only transfected into 293T cells. (C)-(E) shows that Nlrp12 and Hck were co-
transfected into and co-expressed in 293T cells. Panels (A) and (B) are negative controls for
panel (E). Panels (C) and (D) indicate that some cells apparently only express Hck, while some
cells only express Nlrp12 (indicated by triangle arrow). And some cells co-express Nlrp12 and
Hck (indicated by square arrow). Panel (E) indicates that some cells express both Nlrp12 and
Hck, but they are not extensively co-localized.

78
Docking of the Hck 5 amino acids to the Nlrp12’s PYD + NBD domains
A 5 amino acid fragment of Hck Gln507-Ser-Val-Leu-Asp511 was selected for docking
simulations to Nlrp12’s PYD + NBD domains for the following reasons: 1) the docking peptide
requires a short peptide of less than 10 amino acids (Dr. Ian Haworth, USC, personal
communication); and, 2) this 5 amino acid fragment of Hck Gln507-Ser-Val-Leu-Asp511
contained three amino acids (Gln507, Leu510, and Asp511) that were identified as critical amino
acids for binding of Hck to Nlrp12’s PYD + NBD domain. Two potential binding positions were
identified after a trial of 10 docking results (the docking results are ranked from the lowest
energy, i.e., most stable, to the highest energy required for docking) (Figure 11). In the Hck
peptide docking in the left position, there are peptide bonds between Gln of Hck peptide and
manually added -NH2 that are predicted to interact with the -NH and -NH2 group of Arg 358 of
Nlrp12, as well as peptide bond between Gln and Ser in the Hck ligand interacts and the -NH
group in the in the His 189. In contrast, in the Hck peptide docking in the right position, there are
peptide bonds between Ser and Val of the Hck peptide is predicted to interact with Gln265 of
Nlrp12. The -OH group in Ser of the Hck peptide interacts with the -SH group in Cys 262 of
Nlrp12 and –SH- group in methionine (Met) 255 of Nlrp12. The -CO- bond and -NH- bond in
the peptide bond between Ser and Gln of the Hck peptide is predicted to interact with the -OH
group in Ser 258 and -NH2 group in Gln 257 of Nlrp12. Finally, the peptide bond between Gln of
Hck ligand and manually added CH2OH interacts with the -COOH group in Glu 477 of Nlrp12.
The -NH2 group in Gln of the Hck peptide interacts with the -OH group in Tyr 509 of Nlrp12.
There are much fewer electrostatic interactions of the Hck peptide docking in the left position,
indicating that the right position may be more likely to occur. These data may be used to guide
79
further mutagenesis studies applied to the characterization of key structural features of the
binding interaction.

80

81

82

Figure 11: Docking of Hck’s 5 amino acid fragment Gln507-Ser-Val-Leu-Asp511 to
Nlrp12’s PYD + NBD. The structure of Nlrp12’s PYD + NBD was obtained from SWISS-
MODEL website, a homology-modeling server (https://swissmodel.expasy.org/) (Waterhouse et
al., 2018), and based on the published Nlrp3 structure (Sharif et al., 2019). The structure of
Nlrp12’s PYD + NBD was generated based on the homology of amino acids 124-526 in Nlrp12
and Nlrp3. Hck’s 5 amino acid fragment Gln507-Ser-Val-Leu-Asp511 was chosen as the
docking peptide. To make the peptide chain complete and stabilized the peptide chain, the Q side
of Hck ligand is manually added -CH2OH at the peptide bond. The D side of Hck ligand is
manually added -NH2 at the peptide bond. These two molecules were prepared as docking
molecules in “Tools/Surface/Binding analysis/Doc prep” options in the UCSF Chimera program
83
(version 1.13.1), with Nlrp12’s PYD + NBD as a “receptor” and Hck’s 5 amino acids Gln507-
Ser-Val-Leu-Asp511 as a “ligand”. The docking was performed in Tools/Surface/Binding
analysis/AntoDock Vina” options in Chimera. The structure of Hck’s 5 amino acid fragment
Gln507-Ser-Val-Leu-Asp511 was non-fixed, meaning that the structure does not necessarily
maintain its structure as it exists within the structure of full-length Hck. (A). Out of the first 10
possible binding structures, there are 2 positions available, and these two positions are both in
the NBD of Nlrp12’s PYD + NBD. (B) and (C). The two docking positions are shown
individually. In the figures, the blue color is “N”, red color is “O”, and yellow color is “S”. The
Gln507-Ser-Val-Leu-Asp511 peptide is (“ligand”) is shown in green, while the golden color is
the truncated Nlrp12 (“receptor”). All of the amino acids in the receptor that can possibly interact
with Gln507-Ser-Val-Leu-Asp511 are also labeled. The dotted lines are examples electrostatic
interactions with an atom to atom’s distance of < 5 Å). Lys: lysine. Asn: Asparagine.

84
Survival rates of AML in some of acute myeloid leukemia patients are related to Hck
Through the Ocomine and TCGA databases of TCGA provisional, TCGA NEJM 2013, Heusera,
Metzeler 1, Metzeler 2, Raponi data, the relationship between the expression of Hck vs. survival
rates of AML patients as well as expression of Nlrp12 vs. survival rate of AML patients were
analyzed (Figure 12). No significant relationship between AML survival rate and Nlrp12 was
observed (Figure 12A and 12C). However, the provisional data showed that high Hck expression
is significantly associated with a low survival rate of AML patients (p=0.0316, Figure 12B). All
other datasets that are TCGA NEJM 2013, Heusera, Metzeler 1, Metzeler 2, Raponi do not
statistically significant, but only a trend, with high Hck is related to low survival rate of AML
(Figure 12D, 12E, 12F, 12G, and 12H).

85

(A)
(B)
(C)
86
H
(E)
(F)

(D)

87

Figure 12: The red curves mean that Hck is highly expressed. The black lines mean that
Hck expression level is lower. (A) and (C). Online clinical data from TCGA NEJM and TCGA
provisional data appears to show that Nlrp12 has no correlation with AML survival rate. TCGA
NEJM, TCGA provisional, Heusera, Metzeler1, Metzeler2, Raponi data (B, D, E, F, G, H),
showing the relationship between Hck expression and AML survival rate. The conclusion is Hck
had no relationship with AML in TCGA NEJM, Heusera, Metzeler1, Metzeler2, and Raponi
(G)
(H)

88
data. However, the provisional data plotted in (D) suggest that upregulated expression of Hck is
associated with negative outcomes for patients with AML.

89
In blood and marrow samples from patients with AML, Nlrp12 and Hck co-occur and are co-
expressed
The odds ratio test (a statistical tst that defines the likelihood of association of two events)
determines whether two proteins either co-occur or are mutually exclusive in their expression.
The Fisher exact test is a statistical test that uses P-values to determine if two events, such as co-
occurrence or mutual exclusivity, are quantitatively significantly associated. Applying these tests
to TCGA provisional data sets for AML patients (The Cancer Genome Atlas Research Network.,
2013; Cerami et al., 2012a; Gao et al., 2013), Nlrp12 and Nlrp1 are the only two Nlrp family
members that show a statistically significant co-occurrence with Hck (Table 5). However,
Nlrp1’s co-occurrence has a P value of 0.048, while Nlrp12’s co-occurrence has a P value of
<0.001. Therefore, Nlrp12 is most likely the only Nlrp family protein that shows co-occurrence
with Hck in samples from patients with AML.

90

Table 5: Co-occurrence of mRNA expression level of Hck and all of the Nlrp family
proteins (from the provisional data, assessed on July 11 2019)
Hck + Nlrp
family proteins
Co-occurrence (C) or
mutual exclusivity (M)
P-value
Nlrp1 C 0.048
Nlrp2 M 0.546
Nlrp3 C 0.415
Nlrp4 M 0.936
Nlrp5 M 0.877
Nlrp6 M 0.670
Nlrp7 M 0.475
Nlrp8 M 0.877
Nlrp9 M 0.491
Nlrp10 M 0.626
Nlrp11 M 0.233
Nlrp12 C <0.001
Nlrp13 C 1.000
Nlrp14 M 0.509

91
Moreover, in this analysis, Nlrp12 is significantly co-expressed with Hck (Pearson coefficient =
0.80 and Spearman coefficient = 0.73, Pearson and Spearman coefficients show the possibility of
a linear relationship of two factors, Figure 13). This analysis is also consistent with Nlrp12 and
Hck co-occurring in the same patients with AML. However, Nlrp12 was not the only Nlrp
protein that shows significant co-expression with Hck. Nlrp1 and Nlrp3 also showed significant
co-expression with Hck. This can be explained by the fact that co-expression is less stringent
than co-occurrence. In summary, Nlrp12 is the only Nlrp protein that shows co-occurrence with
Hck, and it also shows co-expression with Hck, although it is not the only member of the Nlrp
family to do so. Other data sets, including Hausera data, Metzeler 1 data, Metzeler2 data, and
Raponi data, were not available to evaluate the co-expression of Nlrp12 versus Hck, as these
other data sets did not have information regarding Nlrp12 expression.

92

Figure 13: Nlrp12 is co-expressed with Hck in AML patient samples. Both Nlrp12
expression and Hck expression data are shown as log2 values. The data are RNA seq data
obtained from cBioportal (provisional) database assessed on July 11, 2019. The graph was taken
from the cBioportal website. Spearman coefficient, indicating the association of Nlrp12 and Hck
mRNA expression level (if Nlrp12 and Hck mRNA expression levels are non-parametric) = 0.80.
Pearson coefficient, indicating the degree of the linear relationship between the Nlrp12 mRNA
expression level and Hck mRNA expression level (if Nlrp12 and Hck mRNA expression levels
are parametric) = 0.73. The p-values for Spearman coefficient and Pearson coefficient are both <
0.001.
93
Other potential binding partners of Nlrp12 from the yeast two-hybrid screen
Other potentially interesting and unique genes whose products may interact with Nlrp12’s PYD
+ NBD were identified in the yeast two-hybrid screen (Table 3 and Table 4). Table 4 only shows
those hits with frequency larger than 2, and these hits were analyzed through an IPA diagram
(Figure 14). In the IPA diagram, protein-protein interactions found by our yeast two-hybrid
screen are connected by solid lines and interactions between protein binding partners collated by
IPA are connected by dashed lines. In addition, those molecules that are associated with AML
are colored purple. Interestingly, Nlrp12 is also associated with AML from the database
Catalogue of somatic mutations in cancer (COSMIC). Although the mutation occurs in the NBD
of Nlrp12, only 2 samples of these mutations are observed within 175 samples.

The interaction of one of the putative binding partners to Nlrp12 identified in our screen,
TRAF3IP3 was validated in a co-immunoprecipitation experiment where the FLAG-tagged form
of TRAF3IP3 and V5-tagged form of Nlrp12 were overexpressed in 293T cells. TRAF3IP3 was
coimmunoprecipitated by adding mouse monoclonal anti-TRAF3IP3 antibody, and a band that
showed the same molecular weight size of Nlrp12 was detected by rabbit monoclonal anti-V5
antibody (Figure 14). However, further validation of these other putative interactors detected by
the yeast two-hybrid needs to be done.
94

Figure 14: IPA graph. (A). IPA displaying the 13 distinctive hits from the total results of the
yeast two-hybrid screen for proteins that interacted with Nlrp12. The figure only shows the
Nlrp12-interacting proteins with hits that have a frequency larger than 2. The solid lines indicate
interacting proteins found through our yeast two-hybrid screen. The dashed lines are the Nlrp12-
interacting proteins previously reported in published data. The purple-colored molecules are
those associated with AML. (B). To validate an interaction shown in the IPA diagram,
95
TRAF3IP3-3FLAG and Nlrp12-V5 were co-immunoprecipitated from co-transfected 293T cells.
A non-denaturing cell lysate buffer (see Material and Methods) was used to lyse the cell, and co-
immunoprecipitations were performed with a mouse monoclonal anti-TRAF3IP3 antibody
followed by protein A/G agarose. The immunoprecipitates were analyzed for Nlrp12 by
immunoblotting with rabbit monoclonal anti-V5 antibody. The results from the co-
immunoprecipitation are consistent with a confirmation of an interaction between TRAF3IP3
and Nlrp12.

96
Discussion
In this chapter, the yeast two-hybrid assay was used to screen for Nlrp12 binding partners. This
approach identified 48 genes as positive hits encoding proteins that could possibly bind to
Nlrp12. Among the hits, the Src non-receptor tyrosine kinase family member Hck was selected
to focus upon as a potentially interesting target, particularly given its association with
hematopoiesis and AML. Further direct screens found that Nlrp12 specifically binds to Hck, but
not to Nlrp8 or Nlrp3. Conversely, Hck, but not other members of the Src non-receptor tyrosine
kinase family, specifically binds to Nlrp12. Hck’s C-terminal 30 amino acids are sufficient for
binding to Nlrp12. And, amino acids Phe503, Gln507, Leu510, and Asp511 of Hck are critical
for binding to Nlrp12. Co-immunoprecipitation experiments from mammalian cells confirmed
that Nlrp12 and Hck interact. The docking experiment generated preliminary data regarding the
binding of Hck’s 5 amino acids “Gln507 -Ser -Val- Leu- Asp511” to Nlrp12’s PYD + NBD’s
domains. Finally, a bioinformatics analysis shows that Nlrp12 and Hck essentially exclusively
co-occur, and mRNA levels for both are correlated in blood samples from AML patients.
Together, these data suggested that Nlrp12 and Hck protein and mRNA co-expression may have
an effect on AML.

The binding data from yeast two-hybrid (Figure 7 and Figure 8) showed that the R2 and R3
regions are both important for binding, but we suspect that R3 region is the major region that is
critical for binding, while R2 region may be an “auxiliary” region that is also critical for binding,
because when we delete the R2 region but maintain the R3 region, the R3 region itself cannot
maintain stable binding of Hck’s C-terminal 40 amino acids to Nlrp12’s PYD + NBD. Thus,
binding of Nlrp12’s PYD + NBD to this fragment of Hck may occur initially through binding to
97
R3, followed by stabilization of the interaction by secondarily binding to R2. This model is also
consistent with the finding that when we mutated the R3 region, the binding of Hck’s C-terminal
40 amino acid fragment to the Nlrp12’s PYD + NBD was lost despite the R2 region being
present. The above evidences are consistent with the model that R2 and R3 regions together are
both necessary for binding, but the stabilization of binding to the R3 region requires the presence
of R2.

The docking experiments used a peptide comprised of only five amino acids that are located in
the R3 region in Hck’s C-terminus as the ligand, and Nlrp12’s linker between PYD and NBD
plus NBD as the receptor. In addition, Nlrp12’s truncated structure was obtained through the
homology modeling of the structure with the published structure of Nlrp3 (Sharif et al., 2019).
Thus, although the results are quite preliminary, it is interesting that the 10 most likely docking
results are represented by only two positions.

The yeast two-hybrid screen with Nlrp12 also identified 47 other genes as potential interactors.
According to the Search Tool for Recurring Instances of Neighbouring Genes (STRING) protein
network and previous publications, Nlrp12 interactors include IRAK1 (Williams et al., 2005),
NIK (Lich et al., 2007), TRAF3 (Allen et al., 2012), HSP90 (Arthur et al., 2007), Fas associated
factor 1 (FAF1) (Pinheiro et al., 2011), and apoptosis-associated speck-like protein containing a
caspase activation and recruitment domain (ASC) proteins. IRAK1, NIK, TRAF3, and HSP90
were only found in experiments involving semi-endogenous protein co-immunoprecipitations
with Nlrp12. FAF1 was found to be an interacting protein in NMR experiments by showing a
difference in the chemical shift perturbation of FAF1’s ubiquitin-associated domains (UBA)
98
domain alone and Nlrp12’s PYD domain bound to FAF1’s UBA domain. And FAF1 was also
found to interact with Nlrp12 in a yeast two-hybrid study where the PYD domain of different
Nlrp proteins were screened (Kinoshita et al., 2006). However, in our study, IRAK1 (Shi et al.,
2019), NIK (Fu et al., 2017), TRAF3 (Wang et al., 2018), HSP90 (Kinnaird et al., 2017), FAF1
(Ryu et al., 2003), and ASC (Martin et al., 2016) proteins were not found in our results of
interacting proteins, although they are all expressed in leukocytes. Therefore, given these
differences, the results from this yeast two-hybrid screen may provide a unique database for
future investigations on the proteins that interact with Nlrp12, and the functional outcomes of
these interactions.

Given the interaction that was characterized between Nlrp12 and Hck, we sought to identify
diseases in which their interaction may be relevant. AML is a type of blood and bone marrow
cancer that is characterized by an unusually high level of circulating immature white blood cells
(Prada-Arismendy et al., 2017). In our own analysis, Hck protein expression level is negatively?
related to AML survival rate in some samples from AML patients. This is consistent with the
findings in the literature. For example, Hck is found to be highly expressed in hematopoietic
stem cells of AML patients (Roversi et al., 2017). The overexpression of Hck is linked with the
development of AML (Poh et al., 2015; Roversi et al., 2017). Therefore, Hck may be potentially
used as a blood biomarker to identify those patients with potential to develop of AML disease.
In addition, activation of Hck and its signaling pathways often causes the development of AML
(Dos Santos et al., 2008; Lopez et al., 2016). Hck has been reported to be involved in the FLT3-
HCK-CDK6 pathway. The mutation in FLT3-ITD causes the activation of Hck, and
subsequently results in overexpression of CDK6 in MV4-11 cells, a human AML cell line with
99
the FLT3-ITD mutation. CDK6 siRNA resulted in MV4-11 cells with the FLT3-ITD mutation
having a lower proliferation rate compared to MV4-11 control cells (Lopez et al., 2016). Also, it
has been reported that in AML patients, Hck, Lyn, and Fgr phosphorylation levels are high (Dos
Santos et al., 2008). In summary, the above evidence suggests that either high expression and/or
high activation of Hck could occur in AML patients as well.

Our bioinformatics analysis revealed that Nlrp12 is likely the only member of the Nlrp family
that shows significant co-occurrence with Hck at the level of mRNA expression. However, our
cellular functional study revealed that Nlrp12 protein expression levels decreased when it is co-
expressed with Hck. This discrepancy might be due to Nlrp12’s mRNA expression level not
correlating with its protein expression level (Greenbaum et al., 2003; Liu et al., 2016; Vogel and
Marcotte, 2012). Nevertheless, these data are still support the possibility that Nlrp12 may have
an effect on AML through its binding to Hck. Nlrp12 and Hck co-expression and co-occurrence
may then also be relevant to the prognosis of AML. However, further analyses need to be
performed to evaluate the ability of Nlrp12 and Hck to serve as biomarkers in the prognosis of
AML.

Nlrp12 and Hck co-expression and co-occurrence may indicate that Nlrp12 and Hck mRNA or
protein expression can impact the same pathway or both be regulated by the same molecule. For
example, in the case two molecules exhibiting co-occurrence, they may interact in the same
signaling pathway, for example, Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)
and glycogen synthase kinase 3 beta (GSK3β) showed co-occurrence in the AML provisional
data, and they are in the same signaling pathway (Prochnicki and Latz, 2017). However, further
100
analyses need to be performed to evaluate the ability of Nlrp12 and Hck to serve as biomarkers
in the prognosis of AML.

Although many Hck inhibitors have been developed, they often lack specificity, with Hck
inhibitors also inhibiting other members of the Src non-receptor tyrosine kinase family (Hu et al.,
2004; Naganna et al., 2019; Patel et al., 2017; Pene-Dumitrescu et al., 2008; Roversi et al., 2017;
Saito et al., 2013). If the potential for the development of AML can be decreased when Nlrp12
does not bind to Hck, then an inhibitor to the interaction between Nlrp12 and Hck can be
designed. On the other hand, if the potential for AML is increased when Nlrp12 and Hck do not
bind, then a drug that facilitates Nlrp12 and Hck interaction could be developed. By this
approach, the non-specificity problem with Nlrp inhibitors may still exist but perhaps to a
smaller extent, as Nlrp proteins are less conserved overall compared to the members of the Src
non-receptor tyrosine kinase family. For example, the amino acid identity between Nlrp12’s
PYD + NBD versus another Nlrp PYD + NBD most closely related to Nlrp12, Nlrp3’s PYD +
NBD, is 54%. But, the protein identity between the Hck C1 fragment versus the one with the
closest relationship, the Lyn C1 fragment, is 78%.

In conclusion, this paper reports the preliminary findings of Nlrp12 interacting with Hck and
Nlrp12 co-occurring with Hck in AML patients. The binding data presented here will expand the
database regarding the interactors of Nlrp12, and additional bioinformatic and functional in vitro
and in vivo analyses may shed light on the functional consequence of the interaction of Nlrp12
with Hck.

101
Chapter 5: Characterization of the functional consequences of Nlrp12’s interaction with
Hck

The data in this thesis are the first to demonstrate the functions of an interaction between Nlrp12
and Hck. In this chapter, several hypotheses regarding the functional consequences of the
interaction between Nlrp12 and Hck were generated and tested. Unfortunately, the functional
consequences of Nlrp12 binding to Hck is not clear, and the characterization needs additional
future study, perhaps with an expanded set of tools and/or models. Each hypothesis will be
described in the following text.

Hypothesis 1: Nlrp12 and Csk compete with each other for binding to Hck’s C-terminus to
regulate Hck activity

Introduction
Csk is a kinase that appeared only to phosphorylate members of the Src family of nonreceptor
tyrosine kinases (SFKs) at the negative regulatory tyrosine phosphorylation site Tyr521. For
example, Csk has been reported to inhibit Src activity, which is to phosphorylate paxillin
(Romanova and Mushinski, 2011). Csk’s structure is similar to that of Src family of nonreceptor
tyrosine kinases, in that it also has SH3, SH2, and SH1 domains, but Csk lacks the N-terminal
acylation site, the positive regulatory Tyr site, and the negative regulatory Tyr site that Src
family members have (Ogawa et al., 2002). It has important roles in regulating cell proliferation,
survival, and cell adhesion (Sun et al., 2018). Csk binds to Src at Src’s C-terminus (Levinson et
al., 2008).
102
However, there are reports of other substrates of Csk, such as paxillin (a cytoskeletal protein)
(Turner, 2000), P2X ligand-gated ion channel 3 (P2X3) receptor (which will be activated when
binding to the extracellular ATP) (Jung et al., 2017), activator protein 1 (AP1) (a.k.a. c-Jun)(a
transcription factor that plays important role in stress signaling pathways) (Yarza et al., 2015),
and interestingly, large tumor suppressor kinase (Lats) because it is a serine/threonine kinase
(Stewart et al., 2003). The biological function of phosphorylating these substrates have been
reported as well. For example, Csk can phosphorylate c-Jun, leading to c-Jun’s degradation (Zhu
et al., 2006). And Lats controls cell proliferation, but Csk inhibits cell proliferation by
phosphorylating Lats (Stewart et al., 2003). It is interesting that these proteins are also substrates
of Csk as Csk is thought to phosphorylate only the C-termini of members of the Src family of
non-receptor tyrosine kinases (Okada, 2012).

Nlrp12 appears to bind to Hck at Hck’s C-terminus. As mentioned above, Csk also binds to Src
at Src’s C-terminus (Levinson et al., 2008). Since Hck is highly conserved with Src, relative to
its primary amino acid sequence, and since they both belong to the Src family of non-receptor
tyrosine kinases, we hypothesized that Nlrp12 and Csk may compete with each other for binding
to Hck’s C-terminus to regulate Hck activity (Figure 15).

Results and Discussion
To test the hypothesis, pEFIRES-P-Nlrp12-V5, pEFIRES-P-Hck, and pEFIRES-P-Csk were all
co-transfected into 293T cells to determine whether Hck phosphorylation level changes when
Nlrp12 is present. Rabbit polyclonal anti-p-Tyr527 Src antibody was used to detect the Tyr-
phosphorylation status of Hck. Six trials have been done. However, different results were
103
obtained each time. Shown here is only one of the trials to indicate that the likely reason that the
experiment did not succeed is because the rabbit polyclonal anti-p-Tyr527 Src antibody did not
recognize its target or the overexpression system did not work to induce phosphorylation of Hck
at Tyr521. As shown in Figure 16, an increasing amount of Nlrp12-V5 plasmid (PEFIRES-P-
puro-Nlrp12-V5)(1 µg, 3µg, and 5µg), a fixed amount of Hck plasmid (PEFIRES-P-hygo-Hck)
(1 µg), and a fixed amount of Csk plasmid (PEFIRES-P-puro-Csk,1 µg), and various amounts of
empty vector (PEFIRES-P-puro) to make the total DNA transfected into the cells equal to 7µg
were transfected into, and expressed in, 293T cells. The cell lysate was immunoprecipitated with
mouse monoclonal anti-Hck antibody, and co-immunoprecipitating Nlrp12-V5 was detected by a
mouse monoclonal anti-V5 antibody. Hck and p-Tyr527 Src in the immunoprecipitates was
probed with a mouse monoclonal anti-Hck antibody and rabbit polyclonal anti-p-Tyr527 Src
antibody, respectively. However, no band indicating the phosphorylation of Tyr527 was
observed (Figure 16), which can perhaps be explained by two reasons. One is that the rabbit
polyclonal p-Tyr527 antibody did not work; as indicated in cells expressing Hck and Csk, a
reactive p-Tyr527 band should have been observed. We used the anti-Src Tyr527 antibody
instead of the anti-Hck Tyr521 antibody. Comparing 10 amino acids in the sequence surrounding
Src Tyr527 and Hck Tyr521, a P at the -2 position of Src Tyris an amino acid with very different
chemisry than an S at the -2 position of Hck 521. We suspect that this may be the reason that
rabbit polyclonal p-Tyr527 Src antibody was not reactive in cell lysate samples. The other
possible reason may be that in this overexpression system Hck is constitutively not inhibited
(i.e., the Tyr521 did not need to undergo phosphorylation). However, a positive control for the
anti-active Csk antibody was not included nor was a control to ensure that Csk was active.
Nevertheless, it is inconclusive whether the phosphorylation status of Hck changes when Nlrp12
104
is present compared to that when Nlrp12 is absent, under the conditions in which Csk is also
present. In addition, the 4G10 antibody, used to detect the Tyr-phosphorylation level of the
whole cell lysate, did not detect any change in cellular Tyr-phosphorylation, suggesting that, if
Nlrp12 binds to Hck, resulting in its activation, it is not observed as a global increase in cellular
Tyr-phosphorylation. And Try phosphorylation level of whole cell lysis changed each time out of
6 times (data not shown). The Tyr phosphorylation level changed irregularly when Hck was
overexpressed. This made us to suspect that the subtle changes of experimental conditions can
change the tyrosine phosphorylation level of whole cell lysis.

105

Figure 15: Schematic drawing of the possible relationship between Nlrp12, Csk, and Hck
(Hypothesis 1). Csk and Nlrp12 appear to bind to Hck in the same region (the C-terminus of
Hck). Thus, in the presence of both Csk and Nlrp12, does Hck’s Tyr521 phosphorylation level
(or activation status) change as Csk and Nlrp12 compete for Hck binding? Different colors in
this figure represents the different domains of the proteins. And by binding to Csk, Hck is shifts
from active status (Tyr416 is phosphorylated and Tyr521 is unphosphorylated) to inactive status
(Tyr416 is unphosphorylated and Tyr521 is phosphorylated) (Levinson et al., 2008).

106

Figure 16: Cell lysis and immunoprecipitation of Hck in 293T cells overexpressing Nlrp12-
V5, Hck, and Csk. An increasing amount of Nlrp12-V5 plasmid (PEFIRES-P-puro-Nlrp12-
V5)(1 µg, 3µg, and 5µg), a fixed amount of Hck plasmid (PEFIRES-P-hygo-Hck) (1 µg), a fixed
amount of Csk plasmid (PEFIRES-P-puro-Csk) (1 µg), and various of empty vector (PEFIRES-
P-puro) to make the total DNA transfected into the cells to be 7µg were all co-transfected into
293T cells. The mouse monoclonal anti-Hck antibody, the mouse monoclonal anti-Csk antibody,
and the mouse monoclonal anti-V5 antibody were used to detect the Hck, Csk, and V5 in the cell
107
lysate, individually. The rabbit polyclonal anti-p-Tyr 4G10 antibody was used to detect whole-
cell tyrosine phosphorylation level. The cell lysate was immunoprecipitated with a mouse
monoclonal anti-Hck antibody, and co-immunoprecipitating Nlrp12-V5 was probed by a mouse
monoclonal anti-V5 antibody. Hck was probed by a mouse monoclonal anti-Hck antibody. The
Hck p-Tyr410 band was probed by rabbit polyclonal anti-p-Tyr416 Src antibody. No reactivity
was observed with the rabbit polyclonal anti-p-Tyr521 Src antibody (see text). Because anti-p-
Tyr521 Src antibody did not work or the overexpression system did not work for this experiment,
it is inconclusive whether the phosphorylation status of Hck Tyr521 changes when Nlrp12 is
present compared to that when Nlrp12 is absent, under the conditions in which Csk is also
present. In this experiment, Hck p61 was used instead of Hck p59.

108
Hypothesis 2: The interaction of Nlrp12 with Hck causes or prevents Nlrp12 degradation
and/or Hck degradation.

Introduction
Until now, there have been no publications that have reported that Nlrp12 interacts with other
proteins, thereby causing Nlrp12 degradation. But, a recent publication has reported that
Nlrp12’s interaction with other proteins protects Nlrp12 from degradation. Arthur et al. (2007)
demonstrated that HSP90 interacts with Nlrp12 and thus maintains the stability of Nlrp12 against
degradation. Therefore, when the ansamycin antibiotics geldanamycin
(GA) or radicicol, two
structurally unrelated inhibitors of HSP90’s activities were present in THP-1 monocytic cells,
Nlrp12 underwent degradation through the proteasomal pathway (Arthur et al., 2007).

In addition, interaction of Nlrp12 with other proteins has resulted in the degradation of these
other proteins. For example, Lich et al. (2007) has reported that Nlrp12 stimulated the
degradation of NIK by interacting with NIK when the non-canonical NF-κB pathway was
activated by bacterial lipopeptide Pam3Cys followed by CD40L by stimulation of TLR1 and 2 in
THP-1 cells (Dejardin et al., 2002; Lich et al., 2007). The degradation of NIK was proteasome-
mediated. Normand et al. (2018) reported that Nlrp12 interacts with NOD2. The interaction
promotes the degradation of NOD2 through the K48-linked ubiquitination of NOD2 in the
proteasomal pathway. Nlrp12 also interacts with HSP90. However, when Nlrp12 interacts with
NOD2 instead, this interaction decreases the stability of the NOD2/Receptor-interacting
serine/threonine-protein kinase 2 (RIPK2) protein complex because NOD2 undergoes
ubiquitination and proteasomal degradation. Lowering the stability of the NOD2/RIPK2 complex
109
enhances the response of THP-1 cells to bacterial muramyl dipeptide (MDP), and therefore
tolerance of THP-1 cells to MDP. In Nlrp12 -/- mice, they also found that the absence of Nlrp12
induced the expression of interferon stimulated gene (ISG), and these mice were highly
susceptible to MDP challenge (Normand et al., 2018).

Moreover, Hck itself has been reported to be degraded by casitas B-lineage lymphoma (cbl)
through K63-linked poly-ubiquitination of Hck (Mohapatra et al., 2013). In fact, Cbl is an E3
ubiquitin ligase that is found to ubiquitinate all of the activated protein tyrosine kinases (PTKs)
by directly interacting with activated PTKs to lysosomes or proteasomal degradation (Mohapatra
et al., 2013). Lck, another member of Src family of non-receptor tyrosine kinase, can interact
with HSP90 in vitro (Hartson and Matts, 1994). When stably expressed in Cos-7 cells, Lck
Tyr505Phe, a constitutively active form of Lck,, interacts with HSP90, and Lck Tyr505Phe is
ubiquitinated and undergoes degradation when HSP90 is inhibited by an ansamycin antibiotic
(Giannini and Bijlmakers, 2004; Lu and Hunter, 2009). In addition, Hck can interact with the
ubiquitin E3 ligase and E3 ligase can regulate Hck’s ubiquitination and degradation (Oda et al.,
1999).

All of these data suggest that Nlrp12 and Hck, through their interaction, could potentially alter
each other’s degradation. We hypothesize that the interaction of Nlrp12 with Hck may lead to
either Nlrp12 degradation or Hck degradation. We found that the steady-state expression level of
Nlrp12 decreased when Hck was co-expressed with Nlrp12, observed in three out of three
independent experiments. However, the steady-state expression level of Hck was not observed to
110
decrease when Nlrp12 is co-expressed, suggesting that there is not a reciprocal relationship
between Nlrp12 and Hck, relative to changes in their expression levels.

Results and discussion
Figure 17 shows that in 293T cells, the co-expression of Hck led to a significant decrease in
steady-state protein expression levels of Nlrp12. Although, alternatively, the apparent decrease in
expression level of Nlrp12 might be due actually to a decrease in the epitope-tag itself or a
decrease in the expression of the epitope-tagged protein only. Interestingly, however, Hck
protein levels did not show an increasing level of expression upon transfection with increasing
amounts of plasmid DNA, possibly due to Hck’s maximum expression levels in these cells being
achieved with the lowest amounts of plasmid DNA, which may be resolved if the transfections
are performed with lower amounts of plasmid DNA.

Subsequently, we did another experiment with Nlrp3 was used as a control for Nlrp12, to
determine whether there are differences between the effect of co-expression of Hck resulted in
lower steady-state protein expression levels for Nlrp3 and Nlrp12 (Figure 18A). While the
steady-state expression of Nlrp12 and Nlrp3 both decreased when co-transfected with Hck, there
may be a lower level of Nlrp12, compared to that of Nlrp3 (Figure 18B and 18C). However,
whether a difference exists between Nlrp12 and Nlrp3 expression when co-transfected with Hck
is unknown, since only one experiment was done. In addition, in the overexpression system
where all the Nlrps except Nlrp4 and Hck were transfected into the 293T cells, Nlrp3 interacts
with Hck (Figure 19). In fact, Nlrp1, Nlrp2, Nlrp3, Nlrp5, Nlrp6, Nlrp7, Nlrp8, Nlrp9, Nlrp11,
Nlrp12, Nlrp13, and Nlrp14 interact with Hck (co-transfection of Nlrp4 and Hck was not
111
performed and result on Nlrp10 and Hck interaction is not clear) (Figure 19). But whether Nlrp3
interacts with Hck needs to be confirmed by a stable cell line of Nlrp3 or a stable cell line of Hck
(Jenny P.-Y. Ting, personal communication). Use of the stable line as a method of confirming
the protein-protein interactions are also showed by publications (He et al., 2016; Normand et al.,
2018).

Next, we would like to know if there is a reciprocal relationship between Nlrp12 and Hck.
However, the results do not show any changes in Hck when transfected with theoretically
increased amount of Nlrp12, although the experiments are only done twice (Figure 20). As
showed by Figure 20, Nlrp12 protein expression levels did not increase although Nlrp12 is
transfected with increasing amount. Theoretically, Nlrp12’s amount should increase. The
possible reason is that the expression of Nlrp12 was already at a maximum at the lowest amount
of plasmid used for transfection. Alternatively, the expression level of Nlrp12 protein could be
lowered by the co-expression of Hck. In other words, in the presence of Hck expressed from
transfection with 2.5 µg of Hck expression plasmid, the amount of Nlrp12 expressed from
transfection with 2.5 µg of Nlrp12 expression plasmid may already be at a maximum.
Transfection with larger amounts of Nlrp12 plasmid would thus not result in any higher
expression of Nlrp12 protein.
112

Figure 17: The co-expression of Hck protein significantly decreased the steady-state protein
expression levels of Nlrp12. (A). Epitope-tagged Nlrp12-V5 and Hck p61 were co-transfected
into 293T cells. Cell lysates were immunoblotted with rabbit monoclonal anti-V5 antibody for
Nlrp12 and mouse monoclonal anti-Hck antibody. Comparing lane 2 (basal level of Nlrp12
expression) with lanes 5, 6, and 7, co-expression of Hck protein leads to a significant decrease in
protein expression levels of Nlrp12 (N = 3). Increasing protein expression of Hck, relative to the
amount plasmid DNA added (ramp: lanes 5, 6, and 7; 2.5-20 µg of DNA) was not observed
possibly because the lowest amount of plasmid used for Hck transfection was already too high,
113
resulting in the maximum expression of Hck at all plasmid DNA amounts. The results in this
figure are representative of 3 independent experiments. (B). Quantification of the Nlrp12 protein
bands (mean + standard error) to show that Nlrp12 protein expression levels are decreased in the
presence of co-expressed Hck, relative to equal amounts of cell lysate being assayed (i.e., 20 µg
of cell lysate proteins were loaded for each lane). Nlrp12 protein expression levels are indicated
as arbitrary units. (C). Quantification of the Hck protein bands (mean + standard error)
confirming that Hck protein expression levels did not change although the amount of expression
plasmid DNA for Hck increased. Figure (B) and (C) were generated by Prism 6.0 (GraphPad
Software, Inc., San Diego, CA). ANOVA and Tukey tests were used to compare the Nlrp12 and
Hck protein bands. In this experiment, Hck p61 was used instead of Hck p59.

114

Figure 18: The stability of Nlrp3 vs. Nlrp12 in the presence of increasing amounts of Hck
expression plasmid. (A). (A) is a re-organized version of the original western blot. The reason to
re-organize the blot is that the samples in the original western blot were not correctly
sequentially organized. A 2.5 µg of epitope-tagged Nlrp3-3FLAG or 2.5 µg of epitope-tagged
Nlrp12-3FLAG, and an increasing amount of Hck p61 (2.5 µg, 5 µg, 10 µg, and 20 µg) were co-
transfected into 293T cells. Epitope-tagged Nlrp3-3Flag was used as a control for Nlrp12
transfected with Hck p61. Cell lysates were blotted with a mouse monoclonal anti-FLAG
antibody and a mouse monoclonal anti-Hck antibody. (B). Quantification of the Nlrp12-3FLAG
and Nlrp3-3FLAG protein bands (mean + standard error). The quantification of each band was
relative to equal amounts of cell lysate being assayed (i.e., 20 µg of cell lysate proteins were
loaded for each lane). The Nlrp12-3FLAG and Nlrp3-3FLAG protein expression levels are
115
indicated as arbitrary units. (C). Quantification of the Hck protein bands between the cells co-
transfected with Nlrp12-3FLAG and Nlrp3-3FLAG (mean + standard error). In this experiment,
Hck P61 was used instead of Hck p59.

116

Figure 19: Overexpression of members of the Nlrp family of proteins and Hck in 293T
cells. A 2.5 µg of plasmid DNA encoding different Nlrp family proteins and 2.5 µg of Hck
expression plasmid were co-transfected in 293T cells. Cell lysates were immunoprecipitated with
117
mouse monoclonal anti-Hck antibody, and the Nlrp family proteins that co-immunoprecipitated
with Hck were probed with a mouse monoclonal anti-FLAG antibody (see Material and Methods
for transfection and immunoprecipitation procedures). From the figures, Nlrp1, Nlrp2, Nlrp3,
Nlrp5, Nlrp6, Nlrp7, Nlrp8, Nlrp9, Nlrp11, Nlrp12, Nlrp13, and Nlrp14 can apparently be co-
immunoprecipitated with Hck. Nlrp4 is not included in the overexpression system. The
interaction of Nlrp10 and Hck is not clear because the blot was cut off at 75 KDa, right at the
molecule weight of Nlrp10, 74.9 KDa. The arrows show the Nlrp family proteins and Hck. In
this experiment, Hck P61 was used instead of Hck p59.

118

Figure 20: Co-expression of Hck and Nlrp12 showed no change in Hck expression level,
although the transfected amount of expression plasmid for Nlrp12 was increased. (A).
Epitope-tagged Nlrp12-V5 and Hck p61 were co-transfected into 293T cells. Cell lysates were
blotted with a mouse monoclonal anti-V5 antibody and a mouse monoclonal anti-Hck antibody.
Comparing lane 4 with lanes 5, 6, and 7, Nlrp12 did not show a significant increase in protein
expression level, despite increasing the amount of its expression plasmid. In addition, the amount
of Hck protein did not show any significant changes either. This figure is representative of the
results of two experiments. (B). Quantification of the Nlrp12 protein bands (mean + standard
error) to show that Nlrp12 protein expression levels did not change in the presence of co-
expressed Hck, although the transfected amount increased. The quantification of the bands was
119
relative to equal amounts of cell lysate being assayed (i.e., 20 µg of cell lysate proteins were
loaded for each lane). The Nlrp12 protein expression levels are indicated as arbitrary units. (C).
Quantification of the Hck protein bands (mean + standard error), confirming that Hck protein
expression levels were equal despite differences in the amount of plasmid used for transfections.
Figure (B) and (C) were generated by Prism 6.0 (GraphPad Software, Inc., San Diego, CA).
ANOVA and Tukey tests were used to compare the differences in Nlrp12 and Hck protein bands.
In this experiment, Hck p61 was used instead of Hck p59.

120
Hypothesis 3: Nlrp12 is a p-Tyr substrate of Hck.

Introduction
Hck, as one of the members of the Src family of nonreceptor tyrosine kinases, has many
documented p-Tyr substrates. These p-Tyr substrates of Hck can be seen in Poh, et al.,’s review
(Poh et al., 2015). In addition to the review, two other substrates have been found in the literature
(Table 6). The substrates all have SH3 and/or SH2 domains in common. SH3 and SH2 domains
in these substrates have important roles in the recognition of the substrate by Hck, and aid in
recruiting Hck to the right cellular localization (Briggs et al., 1995; Pellicena et al., 1998). The
SH3 domain binds to proline-rich motifs, so substrates with proline-rich motifs can be
recognized by the SH3 domain of Hck (Briggs et al., 1995). While Nlrp12 does not have an SH3
domain, it has a proline-rich motif, which contains the consensus PxxP sequence, where x
represents any amino acids (Kay et al., 2000) and can potentially bind to SH3 domain-containing
proteins. However, Nlrp12 does not have an SH2 domain, which binds to p-Tyr-containing
proteins that must be present in the sequence pTyr-Glu-Glu-Ile (Nam et al., 2004). In addition,
Src, the founding member of the Src family of nonreceptor tyrosine kinases, prefers the Glu-Glu-
Ile-Tyr-Glu-Glu-Phe consensus sequence in its substrate, although this sequence is not very
stringent (Miller, 2003). But alignment of Nlrp12 from multiple mammalian species (human,
mouse, rat, pig, cow, and chicken) using clast omega software in the website
(https://www.ebi.ac.uk/) revealed that the tyrosine residues Tyr16, Tyr129, Tyr132, Tyr157,
Tyr246, Tyr377, Tyr435, Tyr438, Tyr548, Tyr608, Tyr671, Tyr702, and Tyr1057, are common
among all the species, but confirmed that Nlrp12 does not contain the Glu-Glu-Ile-Tyr-Glu-Glu-
Phe sequence or any sequences that are similar to Glu-Glu-Ile-Tyr-Glu-Glu-Phe sequence
121
(Figure 21). Thus, phosphorylation of Nlrp12 by Hck may involve Tyr-phosphorylation at a
novel site, within the context of an unusual motif. However, overall, it is curious that Nlrp12 is a
substrate of Hck. Nevertheless, here, since Nlrp12 has a Pro-x-x-Pro sequence, we hypothesize
that Nlrp12 may bind to Hck through Hck’s SH3 domain and become a substrate of Hck.
Although the results from the experiment is consistent with the Tyr-phosphorylation of
transfected Nlrp12, the experiment has been not done enough times (i.e., the experiments only
done twice); thus, additional replicates must be performed to confirm the result.

122

Table 6: Substrates of Hck (continue (Poh et al., 2015) with modifications)
Substrates Effects after being phosphorylated citations
Ras GTPase-activating protein (GAP) Unknown
Briggs et al.,
1995
Nef, a HIV protein
1
Unknown
Alvarado et
al., 2014
1. Abbreviation was found in Moarefi et al., 1997.
The other abbreviation was found in uniport.org (The UniProt Consortium, 2019).

123
Mouse MLPSTARDGLYRLSTYLEELEAGELKKFKLFLGIAEDLSQDKIPWGRMEKAGPLEMAQLM 60
Rat MLPATAKDALHRLSTYLEELEAGELKKFKLYLGIAEELGQDKIPRGRMEMAGPLEMAQLM 60
Human MLRTAGRDGLCRLSTYLEELEAVELKKFKLYLGTATELGEGKIPWGSMEKAGPLEMAQLL 60
Chicken MLRTAGRDGLCRLSTYLEELEAVELKKFKLYLGTATELGEGKIPWGCMEKAGPLEMAQLL 60
Pig MSPTPLRNGLCRLCAYLEELEAVELKKFKLYLGMATEMGKHKIPWGRMEPAGPLEMAQLL 60
Cow MSPAPLRSDLGRLCTYLEELEAVELKKFKLYLGTATEMGEGKIPWGRMEQAGPLEMAQLL 60

Mouse VAHMGTREAWLLALSTFQRIHRKDLWERGQGEDLVRVTPNNGLCLF---ESQSACPLDVS 117
Rat VAHMGTKEAWLLALSTFERIHRKDLWERGQGEDLVRVTPSNGLCSL---ESQSTCLSDVS 117
Human ITHFGPEEAWRLALSTFERINRKDLWERGQREDLVRDTPPGGPSSLGN---QSTCLLEVS 117
Chicken ITHFGPEEAWRLALSTFERINRKDLWERGQREDLVRDTPPGGPSSLGNMPSQSTCLLEVS 120
Pig AAHCGIREAWLLTLSIFEQINRKDLWERGQREDLVRETPPGDLFSPGN---QSACSLEDF 117
Cow AAHFGPQEAWLLALSVFHQINRKDLWERGHREDQGKDAPSGGPPSLGS---ESACSLDVL 117

Mouse PNAPRKDLQTTYKDYVRRKFQLMEDRNARLGECVNLSNRYTRLLLVKEHSNPIWTQQKFV 177
Rat PDAPRKDPQITYKDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPIWAQQKLL 177
Human LVTPRKDPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMQVQQQLL 177
Chicken LVTPRKDPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMQAQQQLL 180
Pig PATLRKDPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPLRAQQKLL 177
Cow AAAPRKDPQETYRDYVRRKFRLMEDRNARLGEFVNLSHRYTRLLLAKEHSSPRWAQQKLL 177

Mouse DVEWERSRTRRHQTSPIQMETLFEPDEERPEPPHTVVLQGAAGMGKSMLAHKVMLDWADG 237
Rat ETGWEHSRTRGHQASPIQMETLFEPDEERPEPPRTVVLQGAAGMGKSMLTHKVMLDWADG 237
Human DTGRGHARTVGHQASPIKIETLFEPDEERPEPPRTVVMQGAAGIGKSMLAHKVMLDWADG 237
Chicken DTGRGHARTVGHQASPIKIETLFEPEEERPEPPRTVVMQGAAGIGKSMLAHKVMLDWADG 240
Pig DTGQGHARTVGHPAGLIQMETLFEPDEERPEPPRIVVLQGAAGMGKSMLAHKVMLDWADG 237
Cow DTGRGHAGTAGHPASLIQIEALFEPDEERPEPPRTVVLQGAAGMGKSMLAHKVMLDWADG 237

Mouse RLFQGRFDYVFYISCRELNRSHTQCSVQDLISSCWPERGISLEDLMQAPDRLLFIIDGFD 297
Rat RLFQDQFDYVFYISCRELNRSHTQCSVHDLLSSCWPEHGAPLEDLIRAPDRLLFIIDGFH 297
Human KLFQGRFDYLFYINCREMNQSATECSMQDLIFSCWPEPSAPLQELIRVPERLLFIIDGFD 297
Chicken KLFQGRFDYLFYINCREMNQSATECSMQDLISSCWPEPSAPLQELIRVPERLLFIIDGFD 300
Pig KLFQDRFDYVFYINCREMNQSTVERSAQDLISSCWPEPSVPLHELVRAPERLLFIIDGFD 297
Cow KLFQNRFDYVCYINCREMNRGTAELSVRDLLASCWPEPCAPLQELVRVPERLLVIMDGFD 297

Mouse KLHPSFHDAQGPWCLCWEEKQPTEVLLGSLIRRLLLPQVSLLITTRPCALEKLHGLLEHP 357
Rat ELHPSFHDVQGPWCHCWEEKRPTELLLGSLIRRLLLPQLSLLITTRPCALEKLHGLLEHP 357
Human ELKPSFHDPQGPWCLCWEEKRPTELLLNSLIRKKLLPELSLLITTRPTALEKLHRLLEHP 357
Chicken ELKPSFHDPQGPWCLCWEEKRPTELLLNSLIRKKLLPELSLLITTRPTALEKLHRLLEHP 360
Pig ELKPSFHDPQGPWCLCWEKKRPTEVLLRSLIRKKLMPELSLLITTRPTALEKLHRLLEHP 357
Cow ELRPSFHEPQGPWCLCWEKKAPAEVLLGSLIRKKLLPELSLLITTRPTALAKLQRSLEHP 357

Mouse RHVEILGFSEEARKEYFYRYFHNTGQASRVLSFLMDYEPLFTMCFVPMVSWVVCTCLKQQ 417
Rat RHVEILGFSEAEREEYFYRYFHNTGQASQVFSFMRDYEPLFTMCFVPMVSWVVCTCLKQQ 417
Human RHVEILGFSEAERKEYFYKYFHNAEQAGQVFNYVRDNEPLFTMCFVPLVCWVVCTCLQQQ 417
Chicken RHVEILGFSEAERKEYFYKYFHNAEQADQVFNYVRDNEPLFTMCFVPLVCWVVCTCLQQQ 420
Pig RHVEILGFSEAERKEFFYKYFPSEEQASHVFSFVRDNEPLFTLCFIPMVCWVVCTCLKQQ 417
Cow RHVAILGFSEAERKEYFHRYFHDREQASQVFSFVRDDEPLFTLCFVPMVCWVVCTCLKQQ 417

Mouse LESGELLRQTPRTTTAVYMFYLLSLMQPKPGTPTFKVPANQRGLVSLAAEGLWNQKILFD 477
Rat LESGELLRQTSRTTTAVYMFYLLSLMQPKPGTPTFKVPANQRGLVSLAAEGLWNQKILFE 477
Human LEGGGLLRQTSRTTTAVYMLYLLSLMQPKPGAPRLQPPPNQRGLCSLAADGLWNQKILFE 477
Chicken LEGGGLLRQTSRTTTAVYMLYLLSLMQPKPGAPRLQPPPNQRGLCSLAADGLWNQKILFE 480
Pig LEGGGLLRHTSRTTTAVYLLYLLSLMQPKPGTPTLQPPPNQRGLCSLAADGLWHQKILFE 477
Cow LEGGGLLRRRSRTTTAVYLLYLLSLMQPKPGTPTLPSPPNRRGLCALAAHGLWEQKILFA 477

Mouse EQDLGKHGLDGADVSTFLNVNIFQKGIKCEKFYSFIHLSFQEFFAAMYCALNGRE----- 532
Rat EEDLGKHGLDGAEVSTFLNVNIFQKGIKCEKFYSFIHLSFQEFFTAMYCALHGRE----- 532
Human EQDLRKHGLDGEDVSAFLNMNIFQKDINCERYYSFIHLSFQEFFAAMYYILDEGEGGAGP 537
Chicken EQDLRKHGLDGEDVSAFLNMNIFQKDINCERYYSFIHLSFQEFFAAMYYILDEGERGAGP 540
Pig EQDLRKHGLEVADVSSFLNMNIFQKDINCEKFYSFIHLSFQEFFAAMYYILDGGDSRSGP 537
Cow ERDLREHGLDAADVSSFLNMNIFQKDIICETLYSFIHLSFQEFFAAMHYVLDDGASGSGP 537

Mouse --AVRRALAEYGFSERNFLALTVHFLFGLLNEEMRCYLERNLGWSISPQVKEEVLAWIQN 590
Rat --AVRRALAEYGFSERNFLAHTVRFLFGLLNEEMRCYLERNLGWTISPQVKEEALAWIQN 590
Human DQDVTRLLTEYAFSERSFLALTSRFLFGLLNEETRSHLEKSLCWKVSPHIKMDLLQWIQS 597
Chicken DQDVTRLLTEYAFSERSFLALTSRFLFGLLNEETRSHLEKSLCWKVSPHIKMDLLQWIQS 600
Pig EQNITRLLSEYAFSERSFLALTVRFLFGLLNEETRCSLEKMLGWKVSPHAKMELLEWIRS 597
Cow ERNLSRLLTEYAFSDRSFLALTVRFLFGLLNEETRSYLEKTLGWKVSPGVKTELLEWIRR 597

Mouse KAGSEGSTLQHGSLELLSCLYEVQEEDFIQQALSHFQVVVVRSISTKMEHMVCSFCARYC 650
124
Rat KARSEGSTLQHGSLELLSCLYEIQEEDFIQQALSHFQVVVVRNLSTKMEHVVCSFCARYC 650
Human KAQSDGSTLQQGSLEFFSCLYEIQEEEFIQQALSHFQVIVVSNIASKMEHMVSSFCLKRC 657
Chicken KAQSDGSTLQQGSLEFFSCLYEIQEEEFIQQALSHFQVIVVSNIASKMEHMVSSFCLKHC 660
Pig KAQSEGSTLKQGSLEFFSCLYELQEEEFIQQALSHFQVVVVNSIATKMEHMVSSFCAKNC 657
Cow KARSEGSTLKQGALEVFGCLYELQEEEFIQQALSHFQVVVVNNITTKMEHMVSSFCVKSC 657

Mouse RSTEVLHLHGSAYSTGMEDDPPEPSGVQTQS-TYLQERNMLPDVYSAYLSAAVCTNSNLI 709
Rat RGTEVLHLYGSAYSTGAEDGPPEPPGAQTQS-THSQERNILPDIYSAYLSATICTNSNLI 709
Human RSAQVLHLYGATYSADGEDRARCSAGAHTLLVQLRPERTVLLDAYSEHLAAALCTNPNLI 717
Chicken RSAQVLHLYGATYSADGEDRARCSAGAHTLLVQLRPERTVLLDAYSEHLAAALCTNPNLI 720
Pig RNAAVLQLYGAAYNADGEDGVRWPQ----MPLAQSPERNLLPDAYSEQLAAALSTSPNLV 713
Cow RNAEVLQLFGAAYQAHGEDRLRWP-------LATSPERHVLPDIYSEQLAAALSTNPSLV 710

Mouse ELALYRNALGSQGVRLLCQGLRHASCKLQNLRLKRCQISGSACQDLAAAVIANRNLIRLD 769
Rat ELALYRNALGSQGVRLLCQGLRHANCKLQNLRLKRCHISGSACQDLAAAIIANRNLIRLD 769
Human ELSLYRNALGSRGVKLLCQGLRHPNCKLQNLRLKRCRISSSACEDLSAALIANKNLTRMD 777
Chicken ELSLYRNALGSRGVKLLCQGLRHPNCKLQNLRLKRCRISSSACEDLSAALIANKNLTRMD 780
Pig ELVLYSNALGSRGVKLLCQGLGHPNCNLQNLRLKRCQISSSACQDLALALIANKHLVRMD 773
Cow ELALHSNALGSQGVKLLCQGLRHPNCRLQNLRLKRCQVSSSVCQDLTTALIANKHLLRMD 770

Mouse LSDNSIGVPGLELLCEGLQHPRCRLQMIQLRKCLLEAAAGRSLASVLSNNSYLVELDLTG 829
Rat LSGNSIGVLGLELLCEGLQHPMCRLQMIQLRKCLLEAAAGRALASVLSNNSHLVELDLTG 829
Human LSGNGVGFPGMMLLCEGLRHPQCRLQMIQLRKCQLESGACQEMASVLGTNPHLVELDLTG 837
Chicken LSGNGVGFPGMMLLCEGLRHPHCRLQMIQLRKCQLESGACQEMASVLGTNPHLVELDLTG 840
Pig LSGNSLGLPGVKLLCKGLRHPKCRLQMVQLRKCQLEAGACQEIASVLSSSRHLEELDLTG 833
Cow LSGNALGLLGAQLLCQGLRHPKCRLQVVQLRKCHLEAGACQELASVLSTSRHLLELDLTG 830

Mouse NPLEDSGLKLLCQGLRHPVCRLRTLWLKICHLGQASCEDLASTLKMNQSLLELDLGLNDL 889
Rat NPLEDLGLKLLCQGLRHPVCRLRTLWLKICHLGQASCEDLASTLKMNQSLMELDLGLNDL 889
Human NALEDLGLRLLCQGLRHPVCRLRTLWLKICRLTAAACDELASTLSVNQSLRELDLSLNEL 897
Chicken NALEDLGLRLLCQGLRHPVCRLRTLWLKICCLTAAACDELASTLSVNQSLRELDLSLNEL 900
Pig NALEDSGLRLLCQGLRHPVCRLRILWLKICQLSAAACEDLAATLSGNQSLMELDLSLNEL 893
Cow NALEDSGLRLLCQGLRHPVCRLRILWLKICLLTGAACEDLASTLHMNQSLVELDLSLNDL 890

Mouse GDSGVLLLCEGLSHPDCKLQTLRLGICRLGSVACVGIASVLQVNTCLQELDLSFNDLGDR 949
Rat GDSGALLLCEGLRHPDCKLQTLRLGICRLGSDACAGVASVLQVNTCLRELDLSFNDLGDR 949
Human GDLGVLLLCEGLRHPTCKLQTLRLGICRLGSAACEGLSVVLQANHNLRELDLSFNDLGDW 957
Chicken GDLGVLLLCEGLRHPTCKLQTLRLGICRLGSAACEGLSVVLQANHNLRELDLSFNDLGDW 960
Pig GDPGVLLLCEGLRHPQCKLQTLRLGICRLSSAACEGLSAVLQVNHHLQELDLSFNDLGDC 953
Cow GDPGVLLLCEGLRHPQCKLQTLRLCICRLSSVACEGLSAVLGVSSHLRELDLSFNDLGDR 950

Mouse GLQLLGEGLRHQTCRLQKLWLDNCGLTSKACEDLSSILGISQTLHELYLTNNALGDTGVC 1009
Rat GLWLLGEGLRHQTCRLQKLWLDSCGLTSKACEDLSSVLGISQTLNELYLTNNALGDTGVR 1009
Human GLWLLAEGLQHPACRLQKLWLDSCGLTAKACENLYFTLGINQTLTDLYLTNNALGDTGVR 1017
Chicken GLWLLAEGLQHPTCRLQKLWLDSCGLTAKACKNLYFTLGINQTLTNLYLTNNALGDTGVR 1020
Pig GMSLLCEGLRHPTCRLQKLWLDSCGLTAKACEDLSSMLGVNQTLTELYLTNNVLGNTGVR 1013
Cow GMSLLCEGLRHPTCRLQKL-LDSCSLTGKACEDISSALGINQTLTDLYLTNNALGNTGVR 1009

Mouse LLCKRLRHPGCKLRVLWLFGMDLNKKTHRRMAALRVTKPYLDIGC 1054
Rat LLCKRLRHPGCKLRVLWLFGMDLNKVTHRRMAALRVTKPYLDIGC 1054
Human LLCKRLSHPGCKLRVLWLFGMDLNKMTHSRLAALRVTKPYLDIGC 1062
Chicken LLCKRLSHPGCKLRVLWLFGMDLNRMTHSRLAVLRVTKPYLDIGC 1065
Pig LLCKRLSHPGCKLRVLWLFGMDLNKMTHRSLAALRVTKPYLDIGC 1058
Cow LLCERLSHPGCKLRVLWLFGMDLNKMTHRSLEALRMTKPYLDVGC 1054

Figure 21: The alignment of Nlrp12 from mammalian species showing that tyrosine
residues Tyr16, Tyr129, Tyr132, Tyr157, Tyr246, Tyr377, Tyr435, Tyr438, Tyr548,
Tyr608, Tyr671, Tyr702, and Tyr1057 (these amino acids are underlined and colored red)
are common in all of the species. The alignment was performed using clast omega software at
125
the website (https://www.ebi.ac.uk/). One letter abbreviation of amino acids was used here for
convenience.

126
Results and discussion
Nlrp12-3FLAG and Hck were co-transfected into 293T cells (Figure 22). Nlrp12-3FLAG that
was co-immunoprecipitated after immunoprecipitation of Hck by a mouse monoclonal anti-Hck
antibody was Tyr-phosphorylated that was detected by a mouse monoclonal anti-Tyr
phosphorylation antibody (Figure 22). This experiment was performed twice, with the same
results (i.e, a Tyr phosphorylated band that indicating that Nlrp12 was phosphorylated was
observed after immunoprecipitation of Hck) (data not shown). Therefore, more experiments are
needed to be done to confirm that Nlrp12 is a p-Tyr substrate of Hck.

127

Figure 22: Epitope-tagged Nlrp12 and Hck were co-transfected in 293T cells. Cell lysates
were blotted with mouse monoclonal anti-p-Tyr-100, mouse monoclonal anti-Hck, and mouse
monoclonal anti-FLAG antibodies. Immunoprecipitation of Hck showed that the band of the
same molecular weight of Nlrp12 was co-immunoprecipitated and Tyr-phosphorylated as
detected by mouse monoclonal anti-p-Tyr. This experiment is representative of an experiment
that was performed twice. In this experiment, Hck p61 was used instead of Hck p59.

128
Hypothesis 4: Hck modulates Nlrp12-mediated inhibition of NF-κB activity.

Introduction
The NF-κB pathway (the diagram has been shown in Chapter 1 Figure 1) has been considered to
be prototypical proinflammatory pathway (Dabek et al., 2010; Lawrence, 2009). NF-κB is a
transcriptional factor, which is composed of various numbers of molecules, i.e., the Rel
homology domain (RHD) containing proteins, including RelA (P65), c-Rel, RelB, NF-κB1 (p50
and p105, which is the precursor of p50), NF-κB2 (p52 and p100, which is the precursor of p52)
(Moynagh, 2005). The RHD domain provides a platform to these five parts to form many
homodimers and heterodimers, but not all of them are of biological relevance and are not fully
understood. But, the heterodimers of p65/p50, RelB/p52, RelB/p50, and c-Rel/p50 all clearly
have biological functions (Carlsen et al., 2002; Oeckinghaus and Ghosh, 2009).

Both a canonical pathway and a non-canonical pathway exist for the NF-κB proinflammatory
pathway. In the canonical pathway, the activators are TNF, IL-1 precursor, and TLR (The
UniPort Consortium, 2019). TNF functions as a pro-inflammatory cytokine and can play
multiple roles in cell survival, proliferation, and differentiation (Parameswaran and Patial,
2010). Several TNF family proteins like CD40 can stimulate the non-canonical pathway (see
below) (Hayden and Ghosh, 2014). IL-1 and TLR function as defense against microbial
pathogens (Cohen, 2014; Hug et al., 2018; Poh et al., 2017; Sims and Smith, 2010). Once the
NF-κB pathway is activated, IKK which contains three subunits (α, β and γ) phosphorylates IκB
in the trimer of three subunits: IκB, p65, and P50. Phosphorylated IκB undergoes degradation,
and thus releases p65/p50 subunits. The released P65/P50 subunits enter the nucleus and
129
stimulate the expression of target genes such as cytokines (TNF-α), chemokines (IL-18),
adhesion molecules, enzymes, and other factors (Zhou et al., 2015a).

In the canonical pathway, the activators are lymphotoxin beta (LTβ, which is also known as
TNF-C), CD40 ligand (CD40L, which belongs to TNF family), and B cell-activating factor
(BAFF, which is also featured as a member of the TNF family) (The UniProt Consortium, 2019;
Dejardin et al., 2002; Elgueta et al., 2009). The functions of these three molecules are stimulators
of the pathway. Besides TNF family proteins, non-TNF family proteins and some pathogens can
also stimulate the NF-κB non-canonical pathway. A complete list of stimulators can be found in
Sun’s review (Sun, 2017). Once the NF-κB pathway is activated, the IκB kinase (containing NIK
and two α subunits of IκB) phosphorylates P100 in the P100/RelB dimer. Phosphorylated P100 is
degraded and becomes P52, and at the same time, RelB is released. The RelB/P52 dimer then
enters the nucleus and functions as a transcription factor to stimulate gene expression, such as of
cytokines, some of which are different than those in the canonical pathways (e.g., BAFF),
chemokines that are different than those in the canonical pathways (e.g., B cell attracting
chemokine 1 (BLC, a.k.a., CXCL13), lymphoid organogenesis genes, and other factors (Carlsen
et al., 2002; Lawrence, 2009; Zhou et al., 2015a). A more complete list of the targeted genes of
NF-κB pathway can be found at the website: http://www.bu.edu/nf-kb/gene-resources/target-
genes/. It is generally believed that the canonical NF-κB pathway is involved in innate immunity,
while the non-conical NF-κB pathway is involved in adaptive immunity (Lawrence, 2009).

Previous publications have shown that Nlrp12 inhibits NF-κB pathways (Allen et al., 2012;
Arthur et al., 2007; Arthur et al., 2010; Silveira et al., 2017; Wang et al., 2002; Wang et al.,
130
2018; Zaki et al., 2011)(see Chapter 1 for more information). Ye et al. (Ye et al., 2008) have
published that Nlrp12 interacts with IRAK1 in the canonical pathway. Nlrp12 also interacts with
NIK and TRAF3 in the non-canonical pathway (Allen et al., 2012; Arthur et al., 2007). Please
refer to Chapter 1 for the functions of IRAK1, NIK, and TRAF3. Nevertheless, no matter which
molecules are identified as interacting with Nlrp12 to regulate the NF-κB pathway, Nlrp12
inhibits NF-κB pathways. But, whether Nlrp12 is involved in the canonical NF-κB pathway or
the non-canonical NF-κB pathway is unclear. Although Hck activity is reported not to be
involved in the NF-κB pathway (Smolinska et al., 2011), there is only one publication addressing
this issue. Therefore, we have formally tested the hypothesis that the interaction of Nlrp12 with
Hck relieves the Nlrp12-mediated inhibition of the NF-κB pathways either canonical and/or non-
canonical.

Result and discussion
Cytokine analysis by RT-PCR was performed to test whether Hck has an effect on the Nlrp12
inhibited-canonical NF-κB pathway and/or non-canonical NF-κB pathway under stimulation. To
stimulate the non-canonical pathway in RAW 264.7 cells, 10 µg/µl of lipopolysaccharides (Lps)
was added into the cell culture medium for 0, 1, 3, and 6 hrs. To stimulate the canonical pathway
in RAW 264.7 cells, 1.8 µg/µl of CD40 was added into the cell culture medium for 0, 1, 3, and 6
hrs. Stimulation of the canonical NF-κB pathway or the non-canonical pathway had no apparent
effects on the secretion of cytokines (TNF-α, IL-1β, and IL-6) (Figure 23). Noticeably, there was
no apparent stimulation of the expression of TNF-α, IL-1β, and IL-6 (Figure 23). If stimulation
did occur, then the expected fold changes would be on the order of tens to hundreds compared to
the RAW 264.7 cells that were transfected with empty vector (pEFIRES-P-puro) (Dong et al.,
131
2018). It is possible that the NF-κB pathways were not stimulated due to the selected time points
not being optimal, or there were some technical problems with RT-PCR in faithfully reporting
any changes in expression of the reporter genes.

132

Figure 23: Analysis of cytokine changes by RT-PCR in different stable RAW 264.7 cell
lines (i.e., cells transfected with empty vector (pEFIRES-P-puro); cells transfected with
Nlrp12-3FLAG (pEFIRES-P-puro-Nlrp12-3FLAG); cells transfected with Hck (pEFIRES-
P-hygro-Hck); and cells co-transfected with Nlrp12-3FLAG and Hck (pEFIRES-P-puro-
Nlrp12-3FLAG and pEFIRES-P-hygro-Hck)). (A), (B), and (C). Cells were stimulated with
CD40 (1.8 ug/ml), a known activator of the non-canonical NF-κB pathway. There were no
133
apparent significant changes in cytokines IL-1β, TNF-α, and IL-6 levels among the different cell
lines. Representative results from two experiments are shown. (D). There were no apparent
significant changes in cytokine IL-1β level among the different cell lines, when cells were
stimulated with Lps (10 ug/ml), known to activate the canonical NF-κB pathway. The result of a
single experiment is shown.

134
To dissect the reasons why NF-κB does not appear to be stimulated or whether there was a
technical problem with RT-PCR, we tested whether NF-κB is activated by stimulation of U937
cells with Lps for 4 hrs by comparing the ratio of phospho-IκB-α to IκB-α. Activation of NF-κB
should result in an increase in the ratio of phospho-IκB-α to IκB-α (Figure 24). We did not test
whether NF-κB activity is stimulated by CD40. In addition, if stimulation of NF-κB was
observed (i.e., the positive control worked), then U937 cells that were stably transfected either
with empty vector (pEFIRES-P-puro); Nlrp12-3FLAG (pEFIRES-P-puro-Nlrp12-3FLAG); Hck
(pEFIRES-P-hygro-Hck); or co-transfected with Nlrp12-3FLAG and Hck (pEFIRES-P-puro-
Nlrp12-3FLAG and pEFIRES-P-hygro-Hck) (Figure 24A) were compared in terms of their
stimulation levels using the ratio of phospho-IκB-α to IκB-α. Changes in the ratio of phospho-
IκB-α to IκB-α were monitored in western blots of cell lysates when U937 cells were stimulated
with 10 µg/ml of Lps, 100ng/ml of TLR2 ligand PM3CSK4, or 20 ng/ml of TNF-a for 4 hrs
(Figure 24B and 24C). Form this experiment we conclude two things: 1) The ratio of phospho-
IκB-α to IκB-α in U937 cells that were transfected with vector alone was increased by
stimulation 10 µg/ml of Lps, 100ng/ml of TLR2 ligand PM3CSK4, or 20 ng/ml of TNF-α for 4
hrs, consistent with the activation of the canonical NF-κB pathway in Lps-, PM3CSK4-, and
TNF-α-stimulated U937 cells, although each experiment was only performed once; and, 2) the
experimental procedure is feasible and can be further used. However, since NF-κB can be
activated by 10 µg/ml of Lps according to changes in the ratio of phospho-IκB-α to IκB-α, this
experiment did not address the reason why no stimulation was observed when we performed RT-
PCR. In addition, we did not test whether non-canonical NF-κB pathway is activated or not in
the CD40 stimulated U937 cells or RAW 264.7 cells.

135

136

Figure 24: Activation of the canonical NF-κB pathway by stimulation of pro-monocytic
U937 cells, as assayed by determining the ratio of phospho-IκB-α to IκB-α. Cells were stably
transfected with either empty vector (pEFIRES-P-puro); Nlrp12-3FLAG (pEFIRES-P-puro-
Nlrp12-3FLAG); Hck (pEFIRES-P-hygro-Hck); co-transfected with Nlrp12-3FLAG and Hck
(pEFIRES-P-puro-Nlrp12-3FLAG and pEFIRES-P-hygro-Hck), or U937 cells transiently
transfected with Hck siRNA, and stimulated with 10 µg/ml of Lps, 100 ng/ml of TLR2 ligand
PM3CSK4, and 20 ng/ml of TNF-α for 4 hrs. (A). Results from three different stable cell lines..
In Nlrp12-3FLAG-expressing cells, the pEFIRES-P-Nlrp12-3FLAG-puro plasmid was
transfected into cells, and 2.5 ug/ml puromycin was used for selection of a pooled clonal
137
population of stable transfectants. With Hck cells, the pEFIRES-P-Hck-hygro plasmid was
transfected into cells and selected to produce pooled clonal cells, with 250 ug/ml hygromycin
used for further selection. In the Nlrp12 + Hck cells, the pEFIRES-P-Nlrp12-3Flag-puro and
pEFIRES-P-Hck-hygro were co-transfected into the cells, and 2.5 ug/ml puromycin and 200
ug/ml hygromycin were used for selection. (B). Detection of the stimulation of the canonical NF-
κB pathway in U937 cells by addition of 10 µg/ml of Lps, 100 ng/ml of TLR2 ligand PM3CSK4,
and 20 ng/ml of TNF-α for 4 hrs. Comparing the cells that were transfected with vector with and
without stimulation, NF-κB was clearly activated. “Hck knock down” indicates that the siRNA
to Hck knocked down Hck expression in U937 cells. The Hck knock down was confirmed by
western blot (data not shown). No firm conclusion could be made from these experiments, except
that the approach appears to be feasible. The levels of IκB-a were used as a loading control here.
(C). Quantification of (B) using Fiji software.

138
Since the results from RT-PCR did not detect whether Hck had an effect on the Nlrp12-inhibited
NF-κB pathway, we decided to test whether the functional consequence of NF-κB signaling
pathways by luciferase reporter assays. However, among the three times that the luciferase assay
was performed (Figure 25), luciferase readings of NF-κB signaling in Nlrp12-transfected cells
compared to the vector only-transfected cells was not decreased after NF-κB signaling was
stimulated with 10 ng/ml of TNF-α; thus, the positive control, in which Nlrp12 expression was
expected to inhibit NF-κB signaling that was stimulated with TNF-a (Figure 25A and 25B) did
not work as expected; inhibition of NF-κB signaling activity by expression of Nlrp12 is a result
which is widely published in the literature (see Chapter 1 and introduction of this hypothesis). In
the absence of TNF-α, the cells that were transfected with large amount of DNA (300ng)
together with Hck showed differences compared to either the cells transfected with vector only,
or the cells transfected with small amount of DNA (30ng), or the cells transfected with small
amount of DNA (30ng) together with Hck. However, since the cells transfected with Nlrp12
alone did not show a decrease in NF-κB signaling activity, it calls into question whether the
luciferase assay was the optimal approach to address this question. The expression of Nlrp12 was
confirmed because we used Renilla as the control for different expression of plasmids across the
different groups.

Unexpectedly, from Figure 25 (A), Hck transfection alone caused NF-κB activity to decrease
compared to vector-transfected cells, which is in general the opposite result from that in Figure
24. However, in contrast to these findings, the literature says Hck is not involved in the
regulation NF-κB activity. Consistent with this literature, the results from our experiments, one
showing Hck expression causing NF-κB activity to decrease, while the other showing Hck
139
expression causing NF-κB activity to increase, suggest overall that Hck expression did not
appear to cause any change of NF-κB activity too. In addition, when Nlrp12 and Hck were both
overexpressed, the activation of the NF-κB pathway did not significantly change after
stimulation by TNF-α (Figure 25A and 25B). However, activation of the NF-κB pathway
significantly increased when Nlrp12 was co-expressed at large amount (i.e., 300 ng) with Hck
protein expression. It was expected that NF-κB activity would be inhibited more when Nlrp12
and Hck were both overexpressed, as long as Nlrp12 expression alone inhibited the NF-κB
pathway, and Hck transfection alone did not change the NF-κB pathway.

Overall, the three experiments had no clear conclusion. And further experiments are needed in
order to have a clear conclusion.

140

Figure 25: Duo-Glo luciferase assay to detect whether Hck affected Nlrp12-inhibited NF-
κB activation. (A) and (B). One day before transfection, 293T cells were seeded at a density of 3
´ 10
5
cells in 24-well plate. The next day, the cells were transfected with either 30, 100, or 300
ng of Nlrp12-3FLAG vector (pEFIRES-P-puro-Nlrp12-3FLAG), 100 ng of Hck vector
(C)
141
(pEFIRES-P-hygro-Hck), 100 ng of pGL4.32[lcu2P/NF-κB-RE/Hygro plasmid (the Photinus
pyralis luciferase/NF-κB-RE/hygromycin resistant), 10 ng of pGL4.74[hRluc/TK]
(Renilla reniformis luciferase/Herpes Simplex-thymidine kinase promoter) plasmid, and various
amounts of empty vector (PEFIRES-P-puro) to bring the total amount of DNA added to the cells
to 500 ng. The transfection reagent used was Lipofectamine® 3000. One day after transfection,
10 ng/ml of TNF-α was added to the cells for 6 hrs, and after 6 hrs, NF-κB luciferase readings
were taken. Renilla, which was used as an internal control, was measured following NF-κB
luciferase measurement. 293T cells stimulated with 10 ng/ml of TNF-α. (A) and (B) are two
independent experiments. Note that the magnitude of the values on the y-axis in (A) and (B) are
different, probably because of the difference between the “new” luciferase assay kit and the
“old” one. (C). The procedure is exactly same at (A) and (B) except that 293T cells were not
stimulated with TNF-α. The scale of the y-axis is significantly greater in (A) and (B) versus (C).
(C) has three replicates. In (C), ANOVA tests followed by Tukey tests were performed to
determine whether there was a statistically significant difference between each treatment.

142
Hypothesis 5: Nlrp12 inhibits or activates Hck activation.

Introduction
Hck activity is usually regulated tightly and is typically maintained an inactive state in cells (Poh
et al., 2015). However, Hck can be activated by many proteins, for example, CD45 (Altin and
Sloan, 1997), PNPT6 (a.k.a. SHP-1) (Mizuno et al., 2002), PTP1B, and PTPa when there is some
type of stimulatory event. They all dephosphorylate the regulatory phosphotyrosine residue at
Tyr521 resulting in the autophosphorylation of the positive regulatory tyrosine residue at
Tyr410. In addition, activation of Hck in primary cultured cells and cell lines (e.g., embryonic
stem cells, primary human macrophages, neutrophils, eosinophils, monocytes, pro-monocytic
cells, multiple myeloma cells, the growth factor-dependent murine pro-B-cell line Baf-B03, and
IL- 6- dependent murine plasmocytoma cell line 7TD-1) can be achieved by stimulation with
lipopolysaccharide (Lps) of the TLR-4, IL-2, IL-6, and GM-CSF (Poh et al., 2015). Additionally,
Hck can also be activated by the HIV-NEF) protein that binds tightly with Hck’s SH3 domain
(Poh et al., 2015). Interestingly, Hck is known to be tyrosine phosphorylated, resulting in an
active kinase, in TLR-4 signaling but not with signaling through other TLRs (Chattopadhyay and
Sen, 2014; Smolinska et al., 2011).

However, in addition to the positive regulatory residue Tyr410 and negative regulatory residue
Tyr521, there are seven additional Tyr residues can be phosphorylated according to previous
publications and high throughput proteomic mass spectrometry-based discovery and data on the
phospoSitePlus® website (Hornbeck et al., 2015). For example, Tyr-29 may represent an
additional phosphorylated site that can contribute to the activation of Hck by an unknown
143
mechanism (Johnson et al., 2000). Another example is co-expression of kinase mutated Hck with
mutated Tyr209 (and thus cannot be phosphorylated) at an SH2 residue. The phosphatase CD45
then caused an increase in dephosphorylation of Tyr521 of Hck (Courtney et al., 2017). Hck
Tyr209 is a conserved phosphorylation site across the Src family kinase and its phosphorylation
can be detected in AML (Jin et al., 2015).

Therefore, the hypothesis that Nlrp12, through its interaction with Hck, will increase or decrease
in Hck activity when Hck is stimulated through Lps’ stimulation of the TLR-4 signaling
pathway. We also expect that the interaction of Nlrp12 with Hck will alter the status of
phosphorylation of all Tyr, and specifically, of Tyr410. To test this hypothesis, we activated Hck
in U937 cells by stimulation with Lps to determine whether the total phosphorylation status of
Hck and of Tyr410 changes when Nlrp12 is present. Expression of Nlrp12 appeared to decrease
the tyrosine phosphorylation level of Hck, but the relationship between the expression of Nlrp12
and total phosphorylation status of Hck, as well as the relationship between expression of
phosphor-Tyr410 of Hck is inconclusive. Since both of the experiments were performed less than
three times, further experiments need to be done. Therefore, no conclusions can be currently
made.

Result and discussion
Three independent experiments showed that twice, the presence of Nlrp12 correlated with lower
Tyr-phosphorylation levels in Hck (Figures 26 and 27) and inconclusive results once (Figure 29).
In Figure 26 and 27, two anti-p-Tyr antibodies (anti-p-Tyr-100 and 4G10), were used on western
blots to report all of Hck tyrosine phosphorylation status. After immunoprecipitation of Hck,
144
probing the immunoprecipitated sample with these two antibodies on western blots detected Hck
Tyr-phosphorylation levels. The anti-4G10 and p-Tyr-100 antibodies are known to both detect
all phosphor-tyrosine in the whole cells, but their preferred recognition of phosphorylation
sequences are somewhat different (Tinti et al., 2012). However, despite the differences of the two
antibodies, in Figures 26 and 27, and Figure 28 in which we average the phosphorylated status of
Figures 26 and 27, the ratio of Hck Tyr-phosphorylation to total Hck was decreased in the
immunoprecipitates, although Figure 28 showed a large standard deviation. One reason that the
standard deviation is large could be that the anti-4G10 and p-Tyr-100 antibodies detected
different phosphorylation bands. However, which residue is phosphorylated and how many
residues are phosphorylated are unknown by this experiment. In addition, a mouse monoclonal
anti-IgG antibody was used as a negative control (Figure 27). No Hck band is observed in the
immumoprecipitate using the mouse monoclonal anti-IgG antibody, confirming that Nlrp12 can
be specifically co-immumoprecipitated by immunoprecipitation of Hck. Another time, an
antibody that detected phosphorylation of Tyr410 of all members of the Src family of non-
receptor kinases was used, instead of p-Tyr-100 or 4G10, to try to determine whether Tyr410
was specifically Tyr-phosphorylated. The amino acids surrounding Src Y416 are well conserved
with those surrounding Hck Tyr410. Whole cell lysates were used as a positive control for
binding of the anti-Src Tyr416 antibody. Unfortunately, no conclusion can be made regarding the
quantification of the western blot bands (Figure 29B) because the western blot itself is not
convincing. It appears to have some problems, such as Nlrp12 is somehow not expressed, or the
right-hand side of the blot of the immunoprecipitate looks like the proteins were not well-
transferred.

145

Figure 26: Immunoprecipitation of Hck from U937 cells showed that the phosphorylation
level of Hck in the immunoprecipitates was decreased when Nlrp12 is co-expressed and co-
immunoprecipitated. (A). U937 cells stably expressing either pEFIRES-P-puro vector or
pEFIRES-P-Nlrp12-3FLAG were stimulated with 1µg/ml of PMA for 24 hours. PMA was used
here because it significantly increased the level of Nlrp12 due to its stimulation of the
cytomegalovirus (CMV) promoter in the expression plasmid for Nlrp12. Then the cells were
stimulated with 1 µg/ml of Lps for the indicated times. After stimulation, the cells were lysed and
immunoprecipitated with mouse monoclonal anti-Hck antibody. An antibody that detects p-Tyr-
146
100 was also used to assay the status of Tyr-phosphorylation levels in Hck. For WCL, a mouse
monoclonal anti-FLAG antibody for epitope-tagged Nlrp12-3FLAG and a mouse monoclonal
anti-Hck antibody were used. (B). Quantification of the ratio of phospho-Hck (pHck) expression
levels to total Hck expression levels. Hck Tyr-phosphorylation status appeared to be decreased
in the immunoprecipitates when Nlrp12 is co-expressed and co-immunoprecipitated.
Alternatively, Nlrp12 may preferentially associate with and co-immunoprecipitate with Hck that
is lower in Tyr-phosphorylation levels.

147

Figure 27: Hck phosphorylation is decreased in Hck immunoprecipitates when Nlrp12 is
co-immunoprecipitated. (A). Everything is the same as Figure 12, except two points. One is
that the mouse monoclonal anti 4G10 antibody instead of the mouse monoclonal anti-p-Tyr-100
antibody was used in this experiment. The mouse monoclonal 4G10 antibody and mouse
monoclonal anti-p-Tyr-100 antibody both detect all phospho-Tyr residues. The other is that the
experiment used anti-mouse monoclonal IgG antibody as a negative immunoprecipitation
148
control. No Hck band was detected when anti-mouse monoclonal IgG antibody was used in the
immunoprecipitation. Note that in this figure, the WCL of Nlrp12-3FLAG cell lysate when IgG
was immunopreicipitating antibody was not loaded into the gel. * indicates that the data may not
be reliable due to the apparent presence of a bubble, an artifact the may affect the interpretation
of the western blot. (B). Quantification of the ratio of pHck protein expression levels to Hck
protein expression levels. Hck’s Tyr-phosphorylation status is decreased when Nlrp12 is co-
expressed after treating cells with Lps for 0, 1, 3, and 5 min. Or, as mentioned above, Nlrp12
may preferentially associate with, and co-immunoprecipitate with, Hck that is lower in tyrosine
phosphorylation levels.

149

Figure 28: Quantification of the pHck protein expression levels to Hck protein expression
levels from Figures 26 and 27. Due to the different p-Tyr antibodies used, for each set of
experimental data, we designate the ratio of pHck/Hck as 1.0 in the U937 cells stably transfected
with vector. Hck Tyr-phosphorylation status is decreased in Hck immunoprecipitates when co-
expressed and co-immunoprecipitated with Nlrp12. * indicates that the data may not be reliable
due to the apparent presence of a bubble in the western blot, an artifact that may affect the
interpretation of the data.

150

Figure 29: Immunoprecipitation of Hck from U937 cells treated with 1 µg/ml of Lps. (A).
For the cell lysates, rabbit polyclonal anti-phosphor-Src (pSrc) Tyr416 antibody, mouse
monoclonal anti-Hck antibody, mouse monoclonal anti-FLAG antibody, and mouse monoclonal
anti-GAPDH were used. Immunoprecipitation was performed by the mouse monoclonal anti-
Hck antibody, and the western blot was probed with rabbit polyclonal anti-pSrc Tyr416 antibody
and mouse monoclonal anti-FLAG antibody. The rabbit polyclonal pSrc Tyr416 antibody is
predicted to detect Tyr-phosphorylated Hck Tyr410 as well (the amino acids surrounding Src
151
Y416 are well conserved with those surrounding Hck Tyr410, so, it is speculated that pSrc
Tyr416 antibody may recognize p-Tyr 410 in Hck protein expression bands. Note that the Nlrp12
band is very weak in the western blot of the immunoprecipitate and the WCL. (B). Quantification
of the ratio of the pHck to total Hck protein expression level. Unfortunately, no conclusion can
be made from this figure because the blot itself is not convincing: Nlrp12 does not appear to be
strongly expressed and/or the blot of the immunoprecipitate looks as if the right side of the blot
was not well-transferred. Moreover, this experiment was only performed once, so more replicates
are needed.

152
Hypothesis 6: Nlrp12 and Hck impact proliferation rates in U937 and K562 human
leukemia cell lines

Introduction
The effects of Hck/Lyn overexpression and/or activation are associated with CML progression
(Donato et al., 2003). The overexpression and/or activation of these kinases are activated and are
phosphorylated by Bcr locus fused with Abl (Donato et al., 2003; Pene-Dumitrescu and
Smithgall, 2010; Quintas-Cardama and Cortes, 2009) . In addition, upregulation of Hck/Lyn
protein expression level and/or activation are observed in CML patients that failed treatment by
Gleevec, a tyrosine kinase inhibitor that can be used to treat CML (Donato et al., 2003).
Futhermore, Hck activation is also related to cell transformation in cytokine-independent growth
of fibroblasts in CML pathogenesis, according to previous reports (Klejman et al., 2002;
Lionberger et al., 2000; Pene-Dumitrescu and Smithgall, 2010; Poh et al., 2015; Poincloux et al.,
2007). Finally, overexpression of Hck protects K562 cells from apopotisis induced by imatinib
(Pene-Dumitrescu and Smithgall, 2010). Therefore, our study first focused on the effects of
overexpression of Hck on cell proliferation rate in CML, using the K562 cell line that are defined
as a human erythroleukemia line (Andersson et al., 1979). And in the future, we plan to study
how Hck activation, rather than overexpression, affects cell proliferation.

K562 cells, which do not appear to express endogenously either Nlrp12 or Hck by western blot
(data not shown), were stably transfected with either empty vector (pMSCV-IRES-neo) or a
vector that expresses Hck (pMSCV-IRES-neo-Hck) (a gift from Thomas E. Smithgall lab). The
153
hypothesis is that the proliferation rate is different in K562 cells expressing Hck, compared to
those without Hck, which may then correlate with Hck’s role in CML.

We also sought to test whether there is any difference in proliferation between an Hck-
expressing stable cell line, an Nlrp12-expressing stable cell line, and an Hck + Nlrp12-
expressing stable cell line. So we did the experiments that compared the proliferation rate of
U937 pro-monocytic cells, which is used as AML cell line (Zhou et al., 2015b), which
endogenously express Hck, and which were stably transfected with either Nlrp12-encoding
vectors or empty vectors (pEFIRES-P-puro). The effects of Nlrp12 on cellular proliferation rate
in AML had not previously been tested.

Result and discussion
K562 cells had no apparent difference in proliferation rate when co-transfected with Hck
compared to the cells that were co-transfected with vector only (Figure 30A and 30B). In
addition, no difference in terms of the proliferation rate between the U937 cells that were
transfected with empty vector and Nlrp12 vector was observed (Figure 30C and 30D). The
proliferation rate that were quantitated by cell counts also showed no difference among the U937
cell lines that were transfected with empty vector and Nlrp12 vector (Figure 30E).

154

155
Figure 30: Cell proliferation performed by AlamarBlue® assay and manual cell counting
to compare the proliferation rates of Nlrp12-expressing U937 cells. (A) and (B). The
proliferation rates of K562 stable cell lines transfected with Hck p59 did not significantly differ
from those transfected with the vector (pMSCV-IRES-neo) alone versus Hck (pMSCV-IRES-
Hck-p59-neo). (C), (D), and (E). There is no difference in the proliferation rate of U937 stable
cell lines transfected with either vector (pEFIRES-P-puro) alone or Nlrp12 (pEFIRES-P- puro-
Nlrp12-3FLAG). (C) and (D) were analyzed by the AlamarBlue® assay. (E) was performed by
manual counting cells with a hemocytometer. Each experiment was performed at least three
times. And the data are represented as mean ± standard deviation (i.e., standard deviation from
the biological replicates). The T-test was used to compare the differences between the cells that
are transfected with vector only and that are transfected with Hck or Nlrp12-3FLAG, but no
statistically significant difference was observed. In this experiment, Hck p59 was used instead of
Hck p61.

156
Conclusions for all the six hypotheses
The functional studies described did not reveal any novel functions resulting from Nlrp12
binding to Hck.

157
Chapter 6: Mice work on acute myeloid leukemia

Introduction
AML patients often have lower neutrophil and monocyte counts compared to healthy controls
(Sharif et al., 2019). Total white blood cell counts are often lower in AML patients too (de Jonge
et al., 2011). In Chapter 3, we concluded that Nlrp12 may have an effect on AML through its
interaction with Hck. Based upon these factors, we compared neutrophil and monocyte counts
between WT and Nlrp12 -/- mice.

Results and discussions
There were no differences in the neutrophil and monocyte counts in the blood from WT
versus Nlrp12 -/- mice
Dendritic cells, eosinophil, neutrophils, monocytes, T cells, and B cells were isolated. However,
for the purpose of this experiment, only results of neutrophils CD11b
Hi
Ly-6G
Hi
and monocytes
CD11b
Hi
Ly-6g
low
were shown, and the cell counts (percentage) were analyzed (Figure 31). For
three mice, there were no significant differences in cell counts (percentage) for neutrophils and
monocytes.

The gating strategy for neutrophils and monocytes is shown in Figure 32. However, since there
are only percentage of the cell counts were analyzed, no conclusion about the blood cell types
from WT versus Nlrp12 -/- mice can be made.

158
On the other hand, a previous publication (Arthur et al., 2010) that concluded that no difference
existed among the blood cell types between WT and Nlrp12 -/- mice. These data are apparently
inconsistent with our hypothesis that Nlrp12 may have an effect on AML through its binding to
Hck. However, in our study, actually numbers of different blood cell types and more mice are
needed to examine whether there is a statistically significant difference in blood cell counts
between WT mice and Nlrp12 -/- mice.

159

Figure 31: Comparison of the neutrophil and monocyte counts between WT mice and
Nlrp12 -/- mice. The percentages of neutrophils (A) and monocytes (B) in the total leukocyte
population in WT versus Nlrp12 -/- mice do not show any significant differences. T-tests were
performed for each cell type. The original data were generated by FlowJo (Becton Dickinson &
Company, San Jose, CA), and the figure was generated by Prism 6.0 (San Diego).

160

Figure 32: The gating strategies for neutrophils and monocytes. First, the dead cells are
excluded by size and granularity (Forward Scatter (FSC) and Side Scatter (SSC), respectively).
The doublet cells are excluded by FSC-A (area) and FSC-H (height), leaving only the singlets.
Leukocytes are identified based on the expression of CD45 (this is the population of cells to be
analyzed). The cells are further divided by CD19- and CD3- positive (FITC and SSC), to
differentiate B- and T- cells from non-T- and non-B- cells. Non-T- and non-B- cells were further
differentiated to identify monocytes and neutrophils by CD11b (eFluor 450) and LY-6g (APC),
respectively.

161
Chapter 7: Conclusions

The following conclusions can be made:
1. The portion of Nlrp12 that contains the PYD and NBD domains uniquely binds to the C-
terminal 40 amino acids of Hck in the yeast two-hybrid assay (Figure 6).
2. The last 30 amino acids of Hck is the shortest fragment of Hck that can bind to the
portion of Nlrp12 that contains the PYD and NBD domains in the yeast two-hybrid assay
(Figure 8).
3. Four mutations made in the Hck C-terminus appear to abolish the interaction with Nlrp12
in the yeast two-hybrid assay. Those mutations are: Phe503Ala, Gln507Ala, Leu510Ala,
and Asp511Ala. They all reside in the R3 domain of the C-terminus of Hck (Figure 7).
4. Nlrp12 binds to Hck when they are co-expressed in mammalian cells as they can be co-
immunoprecipitated (Figures 9, 10, 16, 20, 22, 26, and 27).
5. Hck uniquely co-occurred, but is not necessarily uniquely co-expressed with Nlrp12 in
samples from patients with acute myeloid leukemia (Table 5 and Figure 13).

162
Chapter 8: Future Directions

Future direction of each hypothesis is proposed below:
1. Docking experiments
The results from the docking experiments results are preliminary. However, the top 10 results
showed only 2 positions on Nlrp12’s PYD + NBD where the 5 amino acid fragment of Hck,
Gln507-Ser-Val-Lue-Asp511, can dock. Confirmation of this model can be obtained by
crystallizing the structure of Nlrp12. If we get the structure of Nlrp12, then the docking of these
5 amino acid fragments of Hck to Nlrp12’s PYD + NBD domain can be performed again, and
fragment-based design can be used to generate a structure of Hck’s C-terminal 30 amino acid
fragment than can bind to Nlrp12’s PYD + NBD. These results can be further confirmed by co-
crystallization of Nlrp12 and Hck, followed by creating point mutations, typically to disrupt the
interaction. Currently, the potential amino acids that can be considered for mutation are as
follows. The choice of the amino acids is based on amino acid interactions.
Position 1 (Binding Domain 1)
Val 163
Lys 164
Glu 165
His 166
Ser 167
Asn 168
Leu 177
Asp 178
163
Gly 188
His 189
Gln 190
Ala 191
Ser 192
Pro 193
Ile 194
Lys 195
Ile 196
Glu 197
Thr 198
Leu 199
Phe 200
Glu 201
Glu 205
Arg 206
Pro 207
Glu 355
His 356
Pro 357
Arg 358
His 359
Val 360
164
Glu 361
Position 2 (Binding Domain 2)
Glu 254
Met 255
Asn 256
Gln 257
Ser 258
Ala 259
The 260
Cys 262
Ser 263
Met 264
Gln 265
Asp 266
Leu 267
Ile 268
Phe 269
Ser 270
Cys 271
Asn 505
Cys 506
Glu 507
Arg 508
165
Tyr 509

2. Hypothesis 1: Nlrp12 and Csk compete with each other for binding to Hck’s C-terminus
to regulate Hck activity
Further studies can be done using stable cell lines. For example, experiments can be done on
stable cell lines of cells expressing Hck and Csk. When transfected with pEFIRES-P-Nlrp12-
3FLAG or vector as a control, Hck phosphorylation level can be assayed. In addition, we can
buy active Csk protein, since one of the reasons that the experiment did not work is that that
overexpression of Csk might not result in the expression of active Csk. Additionally, an in vitro
assay can be done, i.e., to purify the protein Csk, Hck, and Nlrp12, and in a test tube, to detect
whether Hck kinase activity is changed or not when Nlrp12 is present, under the condition when
Csk is also present. And also, a small molecular inhibitor used as inhibitor of Csk active form
can be introduced into the cell. And a known substrate can be used as a read out for Csk activity
(Courtney et al., 2017).

3. Hypothesis 2: The interaction of Nlrp12 with Hck causes or prevents Nlrp12 degradation
and/or Hck degradation.
How Hck co-expression affects Nlrp12 expression can be further investigated to determine the
functional outcomes of the Nlrp12-Hck interaction relative to protein stability or degradation.
For example, MG-132 and chloroquine, which are a proteasomal inhibitor and an inhibitor of
lysosomal degradation, respectively, can be added to cells in these experiments to determine
whether the decreasing protein expression level of Nlrp12 in the presence of Hck is due to
proteasomal degradation or lysosomal degradation. In addition, an Nlrp12 stable cell line, with a
166
typically lower level of Nlrp12 expression compared to transiently transfected cells, can be used
to test formally whether Nlrp12 protein expression levels are increasingly decreased when Hck is
expression is increased. Moreover, the rate of Nlrp12 degradation, in the absence or presence of
Hck and/or degradation inhibitors, can be studied and compared in pulse-chase assays (Lich et
al., 2007). Conversely, how the co-expression of an increasing amount of Nlrp12 affects Hck
protein expression can be performed essentially in the same way described above.

Additional replicates to determine how Nlrp12 and Nlrp3 expression levels change when co-
transfected with Hck are needed to clarify the current finding-whether there is a statistical
difference in the levels of expression of Nlrp12 and Nlrp3, when Hck is co-expressed. In
addition, again, it is preferable to use semi-endogenous system, i.e., the stable cell lines
expressing Nlrp12 and Nlrp3 but endogenously expressed Hck.

4. Hypothesis 3: Nlrp12 is a p-Tyr substrate of Hck.
Additional experiments can be performed, such as: 1) co-transfection of Nlrp12-3FLAG with
the mutated form of Hck Tyr416Ala, to test that the p-Tyr phosphorylation of Nlrp12 is
dependent upon the expression of active Hck, 2) co-transfection of Hck with Nlrp12-3FLAG in
which each tyrosine residue in Nlrp12 is mutated to an alanine to characterize which Tyr is/are
phosphorylated (Tyr16Ala, Tyr129Ala, Tyr132Ala, Tyr157Ala, Tyr246Ala, Tyr377Ala,
Tyr435Ala, Tyr438Ala, Tyr548Ala, Tyr608Ala, Tyr671Ala, Tyr702Ala, and Tyr1057Ala); and,
3) co-transfection of Nlrp12-3FLAG and Hck, and adding the Hck inhibitor PP3 to inhibit Hck
activity, again to test that the p-Tyr phosphorylation of Nlrp12 is dependent upon the expression
of active Hck. It is predicted that all these three experiments would result in the co-expressed,
167
epitope-tagged Nlrp12-3FLAG not being phosphorylated. Moreover, mass spectrometry can be
performed as well to analyze whether Nlrp12-3FLAG was Tyr-phosphorylated when co-
transfected with Hck, and/or the putative P-Tyr form of Nlrp12 should be re-immunoprecipitated
from the co-immunoprecipitates with anti-Nlrp12 antibodies to confirm that the P-Tyr band is
Nlrp12. Finally, an in vitro Tyr-phosphorylation assay (e.g., purchase active Hck kinase and
purify recombinant Nlrp12 from bacterial or mammalian cells) can be performed to show that
Nlrp12 is a phospho-tyrosine substrate of Hck in vitro. Lastly, stable cell lines of Hck and
Nlrp12 can be made, and then Nlrp12 phosphorylation can be detected in these cell lines as well.

5. Hypothesis 4: Hck modulates Nlrp12-mediated inhibition of NF-κB activity.
Further experiments can be done to test the hypothesis that Hck modulates the Nlrp12-inhibited
NF-κB pathway. Briefly, the RT-PCR experiment (Figure 23) needs to be repeated. The
luciferase assay should be performed with different stimulants, such as first PM3CSK4, and then
CD40 (Ye et al., 2008) to stimulate the non-canonical NF-κB pathway. And use of an enzyme-
linked immunosorbent assay (ELISA) will be considered to confirm the results obtained with the
RT-PCR and luciferase assays. One of the expected results is that all three assays will show that
Hck and Nlrp12 together increase NF-κB pathway activity because Hck interacts with Nlrp12
and causes Nlrp12’s protein expression level to decrease, thus relieving the inhibition of the NF-
κB pathway by Nlrp12. Western blots can also be done to compare NF-κB activation. For
example, the ratio of phospho-IκB-α to IκB-α can be determined in Nlrp12-, Hck-, and Nlrp12 +
Hck- expressing cell lines, compared to vector controls, for stimulation of the canonical pathway,
where it is expected that NF-κB pathway will be downregulated (the ratio of phospho-IκB-α to
IκB-α will decrease in blots of Nlrp12- and Hck- expressing cells). For the non-canonical
168
pathway, the ratio of p100 to p52 in Nlrp12-, Hck-, and Nlrp12 + Hck- expressing cell lines, can
be compared to vector controls. Cell lines like U937 cells can also be used to understand whether
Nlrp12 protein expression level changes with or without Hck. Furthermore, colon tissues treated
with DDS in Hck -/- mice could be dissected and compared for the level of Nlrp12 between wild
type and Hck -/- mice using western blot. One expected result is that the Nlrp12 protein
expression level will increase in Hck -/- mice. This is because Nlrp12 and Hck co-expression in
the heterologous overexpression system results in a decrease of steady-state Nlrp12 protein
levels. Thus, Nlrp12 protein expression in the Hck -/- mice should increase. These tissues can
also be assayed for the activation of NF-κB, using ELISA for phospho-IκB-α protein and IκB-α
protein for the canonical pathway, and p100/p52 antibody for the non-canonical pathway. The
expected results are the NF-κB activation will be less activated in Hck -/- mice. This is because,
as stated above, Nlrp12 protein expression level should increase, and increase Nlrp12 protein
should then decrease NF-κB activation.

6. Hypothesis 5: Nlrp12 inhibits or activates Hck activation.
More replicates where the anti-Tyr phosphorylation antibody and the anti-Src 416 antibody were
used are needed to confirm any conclusions from these experiments. In addition, it would be
better to use only one anti-Tyr phosphorylation antibody to repeat the experiment since different
antibodies may have a preference in terms of recognizing Tyr phosphorylation within the context
of a set of amino acid sequences. Additionally, it would be better to use an anti-Hck Tyr521
antibody along with the anti-Src 416 antibody to address whether Hck activity changes when
Nlrp12 is present. If the presence of, and interaction with, Nlrp12 either increases or decreases
Hck activity, a mutation or mutations in Nlrp12 can be searched and identified by scanning the
databases for Nlrp12 mutations correlated with diseases. If such Nlrp12 mutation(s) are found,
169
then we could potentially use these Nlrp12 mutation(s) as a “controller” of Hck activity. i.e., to
control the Hck activity by introducing these mutations into Nlrp12 and co-expressing the
mutated Nlrp12 with Hck.

7. Hypothesis 6: Nlrp12 and Hck impact proliferation rates in U937 and K562 human
leukemia cell lines
Besides K562 cells, to confirm that the cells with or without Hck exhibit no difference in
proliferation rates, we can in the future measure proliferation in another CML-like cell lines, e.g.,
TK-6 cells. There are also many other CML cell lines (Clarke and Holyoake, 2017).
Furthermore, to determine whether the proliferation rate of cells transfected with Nlrp12 is
different among the cells transfected with Hck, and the cells transfected with Nlrp12 and Hck,
the same kind of cell line should be used. This will answer the question whether interaction of
Nlrp12 and Hck showed difference in proliferation than Nlrp12 alone and/or Hck alone. In
addition, how activation of Hck will be changed on the cells transfected with Nlrp12 and Hck
verses Nlrp12, or verses Hck can be studied through the western blot.

170
Chapter 9: Bibliography

Adams, J.A. (2003). Activation loop phosphorylation and catalysis in protein kinases: is there
functional evidence for the autoinhibitor model? Biochemistry 42, 601-607.
Allen, I.C. (2014). Non-Inflammasome Forming NLRs in Inflammation and Tumorigenesis.
Frontiers in immunology 5, 169-169.
Allen, I.C., Wilson, J.E., Schneider, M., Lich, J.D., Roberts, R.A., Arthur, J.C., Woodford, R.M.,
Davis, B.K., Uronis, J.M., Herfarth, H.H., et al. (2012). NLRP12 suppresses colon inflammation
and tumorigenesis through the negative regulation of noncanonical NF-kappaB signaling.
Immunity 36, 742-754.
Altin, J.G., and Sloan, E.K. (1997). The role of CD45 and CD45-associated molecules in T cell
activation. Immunol Cell Biol 75, 430-445.
Alvarado, J.J., Tarafdar, S., Yeh, J.I., and Smithgall, T.E. (2014). Interaction with the Src
homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for
kinase activation and effector recruitment. J Biol Chem 289, 28539-28553.
Andersson, L.C., Nilsson, K., and Gahmberg, C.G. (1979). K562--a human erythroleukemic cell
line. Int J Cancer 23, 143-147.
Arthur, J.C., Lich, J.D., Aziz, R.K., Kotb, M., and Ting, J.P. (2007). Heat shock protein 90
associates with monarch-1 and regulates its ability to promote degradation of NF-kappaB-
inducing kinase. J Immunol 179, 6291-6296.
Arthur, J.C., Lich, J.D., Ye, Z., Allen, I.C., Gris, D., Wilson, J.E., Schneider, M., Roney, K.E.,
O'Connor, B.P., Moore, C.B., et al. (2010). Cutting edge: NLRP12 controls dendritic and
myeloid cell migration to affect contact hypersensitivity. J Immunol 185, 4515-4519.
Ataide, M.A., Andrade, W.A., Zamboni, D.S., Wang, D., Souza Mdo, C., Franklin, B.S., Elian,
S., Martins, F.S., Pereira, D., Reed, G., et al. (2014). Malaria-induced NLRP12/NLRP3-
dependent caspase-1 activation mediates inflammation and hypersensitivity to bacterial
superinfection. PLoS Pathog 10, e1003885.
Boggon, T.J., and Eck, M.J. (2004). Structure and regulation of Src family kinases. Oncogene
23, 7918-7927.
Borghini, S., Tassi, S., Chiesa, S., Caroli, F., Carta, S., Caorsi, R., Fiore, M., Delfino, L.,
Lasiglie, D., Ferraris, C., et al. (2011). Clinical presentation and pathogenesis of cold-induced
autoinflammatory disease in a family with recurrence of an NLRP12 mutation. Arthritis Rheum
63, 830-839.
171
Brakke, M.K. (1951). Density Gradient Centrifugation: A New Separation Technique1. Journal
of the American Chemical Society 73, 1847-1848.
Briggs, S.D., Bryant, S.S., Jove, R., Sanderson, S.D., and Smithgall, T.E. (1995). The Ras
GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-
related tyrosine kinase, Hck. J Biol Chem 270, 14718-14724.
Cai, S., Batra, S., Del Piero, F., and Jeyaseelan, S. (2016). NLRP12 modulates host defense
through IL-17A-CXCL1 axis. Mucosal Immunol 9, 503-514.
Carlsen, H.S., Baekkevold, E.S., Johansen, F.E., Haraldsen, G., and Brandtzaeg, P. (2002). B cell
attracting chemokine 1 (CXCL13) and its receptor CXCR5 are expressed in normal and aberrant
gut associated lymphoid tissue. Gut 51, 364-371.
Carreno, S., Gouze, M.E., Schaak, S., Emorine, L.J., and Maridonneau-Parini, I. (2000). Lack of
palmitoylation redirects p59Hck from the plasma membrane to p61Hck-positive lysosomes. J
Biol Chem 275, 36223-36229.
Cecrdlova, E., Petrickova, K., Kolesar, L., Petricek, M., Sekerkova, A., Svachova, V., and Striz,
I. (2016). Manumycin A downregulates release of proinflammatory cytokines from TNF alpha
stimulated human monocytes. Immunol Lett 169, 8-14.
Cerami, E., Gao, J., Dogrusoz, U., Gross, B.E., Sumer, S.O., Aksoy, B.A., Jacobsen, A., Byrne,
C.J., Heuer, M.L., Larsson, E., et al. (2012a). The cBio Cancer Genomics Portal: An Open
Platform for Exploring Multidimensional Cancer Genomics Data. Cancer Discovery 2, 401.
Cerami, E., Gao, J., Dogrusoz, U., Gross, B.E., Sumer, S.O., Aksoy, B.A., Jacobsen, A., Byrne,
C.J., Heuer, M.L., Larsson, E., et al. (2012b). The cBio cancer genomics portal: an open
platform for exploring multidimensional cancer genomics data. Cancer Discov 2, 401-404.
Chattopadhyay, S., and Sen, G.C. (2014). Tyrosine phosphorylation in Toll-like receptor
signaling. Cytokine & growth factor reviews 25, 533-541.
Chen, D.Y., Chen, Y.M., Chen, H.H., Hsieh, C.W., Gung, N.R., Hung, W.T., Tzang, B.S., and
Hsu, T.C. (2018). Human parvovirus B19 nonstructural protein NS1 activates NLRP3
inflammasome signaling in adultonset Still's disease. Mol Med Rep 17, 3364-3371.
Chen, L., Wilson, J.E., Koenigsknecht, M.J., Chou, W.C., Montgomery, S.A., Truax, A.D.,
Brickey, W.J., Packey, C.D., Maharshak, N., Matsushima, G.K., et al. (2017). NLRP12
attenuates colon inflammation by maintaining colonic microbial diversity and promoting
protective commensal bacterial growth. Nat Immunol 18, 541-551.
Chen, S.T., Chen, L., Lin, D.S., Chen, S.Y., Tsao, Y.P., Guo, H., Li, F.J., Tseng, W.T., Tam,
J.W., Chao, C.W., et al. (2019). NLRP12 Regulates Anti-viral RIG-I Activation via Interaction
with TRIM25. Cell Host Microbe 25, 602-616.e607.
172
Clarke, C.J., and Holyoake, T.L. (2017). Preclinical approaches in chronic myeloid leukemia:
from cells to systems. Exp Hematol 47, 13-23.
Cohen, P. (2014). The TLR and IL-1 signalling network at a glance. Journal of cell science 127,
2383-2390.
Courtney, A.H., Amacher, J.F., Kadlecek, T.A., Mollenauer, M.N., Au-Yeung, B.B., Kuriyan, J.,
and Weiss, A. (2017). A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by
CD45. Mol Cell 67, 498-511.e496.
Coutermarsh-Ott, S., Eden, K., and Allen, I.C. (2016). Beyond the inflammasome: regulatory
NOD-like receptor modulation of the host immune response following virus exposure. J Gen
Virol 97, 825-838.
Cowan, E.P., Alexander, R.K., Daniel, S., Kashanchi, F., and Brady, J.N. (1997). Induction of
tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic
virus type 1 Tax. J Virol 71, 6982-6989.
Dabek, J., Kulach, A., and Gasior, Z. (2010). Nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-kappaB): a new potential therapeutic target in atherosclerosis? Pharmacol
Rep 62, 778-783.
Damiano, J.S., Oliveira, V., Welsh, K., and Reed, J.C. (2004). Heterotypic interactions among
NACHT domains: implications for regulation of innate immune responses. The Biochemical
journal 381, 213-219.
de Jonge, H.J.M., Valk, P.J.M., de Bont, E.S.J.M., Schuringa, J.J., Ossenkoppele, G., Vellenga,
E., and Huls, G. (2011). Prognostic impact of white blood cell count in intermediate risk acute
myeloid leukemia: relevance of mutated NPM1 and FLT3-ITD. Haematologica 96, 1310-1317.
Dejardin, E., Droin, N.M., Delhase, M., Haas, E., Cao, Y., Makris, C., Li, Z.W., Karin, M.,
Ware, C.F., and Green, D.R. (2002). The lymphotoxin-beta receptor induces different patterns of
gene expression via two NF-kappaB pathways. Immunity 17, 525-535.
Donato, N.J., Wu, J.Y., Stapley, J., Gallick, G., Lin, H., Arlinghaus, R., and Talpaz, M. (2003).
BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia
cells selected for resistance to STI571. Blood 101, 690-698.
Dong, J., Li, J., Cui, L., Wang, Y., Lin, J., Qu, Y., and Wang, H. (2018). Cortisol modulates
inflammatory responses in LPS-stimulated RAW264.7 cells via the NF-κB and MAPK
pathways. BMC veterinary research 14, 30-30.
Dos Santos, C., Demur, C., Bardet, V., Prade-Houdellier, N., Payrastre, B., and Récher, C.
(2008). A critical role for Lyn in acute myeloid leukemia. Blood 111, 2269.
173
Elgueta, R., Benson, M.J., de Vries, V.C., Wasiuk, A., Guo, Y., and Noelle, R.J. (2009).
Molecular mechanism and function of CD40/CD40L engagement in the immune system.
Immunol Rev 229, 152-172.
Faustin, B., Lartigue, L., Bruey, J.M., Luciano, F., Sergienko, E., Bailly-Maitre, B., Volkmann,
N., Hanein, D., Rouiller, I., and Reed, J.C. (2007). Reconstituted NALP1 inflammasome reveals
two-step mechanism of caspase-1 activation. Mol Cell 25, 713-724.
Fogli, M., Caccuri, F., Iaria, M.L., Giagulli, C., Corbellini, S., Campilongo, F., Caruso, A., and
Fiorentini, S. (2014). The immunomodulatory molecule pidotimod induces the expression of the
NOD-like receptor NLRP12 and attenuates TLR-induced inflammation. J Biol Regul Homeost
Agents 28, 753-766.
Fu, G., Xu, Q., Qiu, Y., Jin, X., Xu, T., Dong, S., Wang, J., Ke, Y., Hu, H., Cao, X., et al.
(2017). Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1. J Exp
Med 214, 1453-1469.
Gao, J., Aksoy, B.A., Dogrusoz, U., Dresdner, G., Gross, B., Sumer, S.O., Sun, Y., Jacobsen, A.,
Sinha, R., Larsson, E., et al. (2013). Integrative analysis of complex cancer genomics and
clinical profiles using the cBioPortal. Sci Signal 6, pl1.
Gharagozloo, M., Mahmoud, S., Simard, C., Mahvelati, T.M., Amrani, A., and Gris, D. (2018).
The Dual Immunoregulatory function of Nlrp12 in T Cell-Mediated Immune Response: Lessons
from Experimental Autoimmune Encephalomyelitis. Cells 7.
Gharagozloo, M., Mahvelati, T.M., Imbeault, E., Gris, P., Zerif, E., Bobbala, D., Ilangumaran,
S., Amrani, A., and Gris, D. (2015). The nod-like receptor, Nlrp12, plays an anti-inflammatory
role in experimental autoimmune encephalomyelitis. J Neuroinflammation 12, 198.
Giannini, A., and Bijlmakers, M.-J. (2004). Regulation of the Src Family Kinase Lck by Hsp90
and Ubiquitination. Molecular and Cellular Biology 24, 5667-5676.
Greenbaum, D., Colangelo, C., Williams, K., and Gerstein, M. (2003). Comparing protein
abundance and mRNA expression levels on a genomic scale. Genome Biol 4, 117.
Guiet, R., Poincloux, R., Castandet, J., Marois, L., Labrousse, A., Le Cabec, V., and
Maridonneau-Parini, I. (2008). Hematopoietic cell kinase (Hck) isoforms and phagocyte duties -
from signaling and actin reorganization to migration and phagocytosis. Eur J Cell Biol 87, 527-
542.
Hafner-Bratkovič, I., Sušjan, P., Lainšček, D., Tapia-Abellán, A., Cerović, K., Kadunc, L.,
Angosto-Bazarra, D., Pelegrin, P., and Jerala, R. (2018). NLRP3 lacking the leucine-rich repeat
domain can be fully activated via the canonical inflammasome pathway. Nature Communications
9, 5182.
174
Hartson, S.D., and Matts, R.L. (1994). Association of Hsp90 with cellular Src-family kinases in a
cell-free system correlates with altered kinase structure and function. Biochemistry 33, 8912-
8920.
Hayden, M.S., and Ghosh, S. (2014). Regulation of NF-κB by TNF family cytokines. Seminars
in immunology 26, 253-266.
He, Y., Zeng, M.Y., Yang, D., Motro, B., and Nunez, G. (2016). NEK7 is an essential mediator
of NLRP3 activation downstream of potassium efflux. Nature 530, 354-357.
Hobbs, S., Jitrapakdee, S., and Wallace, J.C. (1998). Development of a Bicistronic Vector
Driven by the Human Polypeptide Chain Elongation Factor 1α Promoter for Creation of Stable
Mammalian Cell Lines That Express Very High Levels of Recombinant Proteins. Biochemical
and Biophysical Research Communications 252, 368-372.
Hornbeck, P.V., Zhang, B., Murray, B., Kornhauser, J.M., Latham, V., and Skrzypek, E. (2015).
PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 43, D512-520.
Hornick, E.E., Banoth, B., Miller, A.M., Zacharias, Z.R., Jain, N., Wilson, M.E., Gibson-Corley,
K.N., Legge, K.L., Bishop, G.A., Sutterwala, F.S., et al. (2018). Nlrp12 Mediates Adverse
Neutrophil Recruitment during Influenza Virus Infection. J Immunol 200, 1188-1197.
Hoshino, K., Quintas-Cardama, A., Yang, H., Sanchez-Gonzalez, B., and Garcia-Manero, G.
(2007). Aberrant DNA methylation of the Src kinase Hck, but not of Lyn, in Philadelphia
chromosome negative acute lymphocytic leukemia. Leukemia 21, 906-911.
Hu, Y., Liu, Y., Pelletier, S., Buchdunger, E., Warmuth, M., Fabbro, D., Hallek, M., Van Etten,
R.A., and Li, S. (2004). Requirement of Src kinases Lyn, Hck and Fgr for BCR-ABL1-induced
B-lymphoblastic leukemia but not chronic myeloid leukemia. Nat Genet 36, 453-461.
Hug, H., Mohajeri, M.H., and La Fata, G. (2018). Toll-Like Receptors: Regulators of the
Immune Response in the Human Gut. Nutrients 10.
Jacobs, S.R., and Damania, B. (2012). NLRs, inflammasomes, and viral infection. J Leukoc Biol
92, 469-477.
Janowski, A.M., Kolb, R., Zhang, W., and Sutterwala, F.S. (2013). Beneficial and Detrimental
Roles of NLRs in Carcinogenesis. Frontiers in immunology 4, 370-370.
Jeru, I., Duquesnoy, P., Fernandes-Alnemri, T., Cochet, E., Yu, J.W., Lackmy-Port-Lis, M.,
Grimprel, E., Landman-Parker, J., Hentgen, V., Marlin, S., et al. (2008). Mutations in NALP12
cause hereditary periodic fever syndromes. Proc Natl Acad Sci U S A 105, 1614-1619.
Jeru, I., Le Borgne, G., Cochet, E., Hayrapetyan, H., Duquesnoy, P., Grateau, G., Morali, A.,
Sarkisian, T., and Amselem, S. (2011). Identification and functional consequences of a recurrent
NLRP12 missense mutation in periodic fever syndromes. Arthritis Rheum 63, 1459-1464.
175
Jin, L.L., Wybenga-Groot, L.E., Tong, J., Taylor, P., Minden, M.D., Trudel, S., McGlade, C.J.,
and Moran, M.F. (2015). Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain
modulates its binding affinity and specificity. Molecular & cellular proteomics : MCP 14, 695-
706.
Johnson, T.M., Williamson, N.A., Scholz, G., Jaworowski, A., Wettenhall, R.E., Dunn, A.R.,
and Cheng, H.C. (2000). Modulation of the catalytic activity of the Src family tyrosine kinase
Hck by autophosphorylation at a novel site in the unique domain. J Biol Chem 275, 33353-
33364.
Jung, Y.H., Kim, Y.O., Lin, H., Cho, J.H., Park, J.H., Lee, S.D., Bae, J., Kang, K.M., Kim, Y.G.,
Pae, A.N., et al. (2017). Discovery of Potent Antiallodynic Agents for Neuropathic Pain
Targeting P2X3 Receptors. ACS Chem Neurosci 8, 1465-1478.
Kanneganti, T.D., Lamkanfi, M., and Nunez, G. (2007). Intracellular NOD-like receptors in host
defense and disease. Immunity 27, 549-559.
Karan, D., Tawfik, O., and Dubey, S. (2017). Expression analysis of inflammasome sensors and
implication of NLRP12 inflammasome in prostate cancer. Sci Rep 7, 4378.
KAY, B.K., WILLIAMSON, M.P., and SUDOL, M. (2000). The importance of being proline:
the interaction of proline-rich motifs in signaling proteins with their cognate domains. The
FASEB Journal 14, 231-241.
Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H., Zahler, A.M., and Haussler,
D. (2002). The human genome browser at UCSC. Genome Res 12, 996-1006.
Kim, L.C., Song, L., and Haura, E.B. (2009). Src kinases as therapeutic targets for cancer.
Nature Reviews Clinical Oncology 6, 587.
Kinnaird, J.H., Singh, M., Gillan, V., Weir, W., Calder, E.D., Hostettler, I., Tatu, U., Devaney,
E., and Shiels, B.R. (2017). Characterization of HSP90 isoforms in transformed bovine
leukocytes infected with Theileria annulata. Cell Microbiol 19.
Kinoshita, T., Kondoh, C., Hasegawa, M., Imamura, R., and Suda, T. (2006). Fas-associated
factor 1 is a negative regulator of PYRIN-containing Apaf-1-like protein 1. Int Immunol 18,
1701-1706.
Klejman, A., Schreiner, S.J., Nieborowska-Skorska, M., Slupianek, A., Wilson, M., Smithgall,
T.E., and Skorski, T. (2002). The Src family kinase Hck couples BCR/ABL to STAT5 activation
in myeloid leukemia cells. The EMBO journal 21, 5766-5774.
Krauss, J.L., Zeng, R., Hickman-Brecks, C.L., Wilson, J.E., Ting, J.P., and Novack, D.V. (2015).
NLRP12 provides a critical checkpoint for osteoclast differentiation. Proc Natl Acad Sci U S A
112, 10455-10460.
176
Lawrence, T. (2009). The nuclear factor NF-κB pathway in inflammation. Cold Spring Harbor
perspectives in biology 1, a001651.
Lerner, E.C., and Smithgall, T.E. (2002). SH3-dependent stimulation of Src-family kinase
autophosphorylation without tail release from the SH2 domain in vivo. Nat Struct Biol 9, 365-
369.
Levinson, N.M., Seeliger, M.A., Cole, P.A., and Kuriyan, J. (2008). Structural basis for the
recognition of c-Src by its inactivator Csk. Cell 134, 124-134.
Li, J., Moran, T., Swanson, E., Julian, C., Harris, J., Bonen, D.K., Hedl, M., Nicolae, D.L.,
Abraham, C., and Cho, J.H. (2004). Regulation of IL-8 and IL-1beta expression in Crohn's
disease associated NOD2/CARD15 mutations. Hum Mol Genet 13, 1715-1725.
Lich, J.D., and Ting, J.P. (2007). Monarch-1/PYPAF7 and other CATERPILLER (CLR, NOD,
NLR) proteins with negative regulatory functions. Microbes Infect 9, 672-676.
Lich, J.D., Williams, K.L., Moore, C.B., Arthur, J.C., Davis, B.K., Taxman, D.J., and Ting, J.P.
(2007). Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent
chemokine expression in monocytes. J Immunol 178, 1256-1260.
Linz, B.M., Neely, C.J., Kartchner, L.B., Mendoza, A.E., Khoury, A.L., Truax, A., Sempowski,
G., Eitas, T., Brickey, J., Ting, J.P., et al. (2017). Innate Immune Cell Recovery Is Positively
Regulated by NLRP12 during Emergency Hematopoiesis. J Immunol 198, 2426-2433.
Lionberger, J.M., Wilson, M.B., and Smithgall, T.E. (2000). Transformation of myeloid
leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck. J
Biol Chem 275, 18581-18585.
Liu, R., Truax, A.D., Chen, L., Hu, P., Li, Z., Chen, J., Song, C., and Ting, J.P. (2015).
Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer:
correlation with cancer stages and inflammasome components. Oncotarget 6, 33456-33469.
Liu, S., Chen, S., Li, X., Wu, S., Zhang, Q., Jin, Q., Hu, L., Zhou, R., Yu, Z., Meng, F., et al.
(2017). Lck/Hck/Fgr-Mediated Tyrosine Phosphorylation Negatively Regulates TBK1 to
Restrain Innate Antiviral Responses. Cell Host Microbe 21, 754-768.e755.
Liu, Y., Beyer, A., and Aebersold, R. (2016). On the Dependency of Cellular Protein Levels on
mRNA Abundance. Cell 165, 535-550.
Lopez, S., Voisset, E., Tisserand, J.C., Mosca, C., Prebet, T., Santamaria, D., Dubreuil, P., and
De Sepulveda, P. (2016). An essential pathway links FLT3-ITD, HCK and CDK6 in acute
myeloid leukemia. Oncotarget 7, 51163-51173.
Lu, Z., and Hunter, T. (2009). Degradation of activated protein kinases by ubiquitination. Annu
Rev Biochem 78, 435-475.
177
Lukens, J.R., Gurung, P., Shaw, P.J., Barr, M.J., Zaki, M.H., Brown, S.A., Vogel, P., Chi, H.,
and Kanneganti, T.D. (2015). The NLRP12 Sensor Negatively Regulates Autoinflammatory
Disease by Modulating Interleukin-4 Production in T Cells. Immunity 42, 654-664.
Macaluso, F., Nothnagel, M., Parwez, Q., Petrasch-Parwez, E., Bechara, F.G., Epplen, J.T., and
Hoffjan, S. (2007). Polymorphisms in NACHT-LRR (NLR) genes in atopic dermatitis. Exp
Dermatol 16, 692-698.
Martin, B.N., Wang, C., Zhang, C.J., Kang, Z., Gulen, M.F., Zepp, J.A., Zhao, J., Bian, G., Do,
J.S., Min, B., et al. (2016). T cell-intrinsic ASC critically promotes T(H)17-mediated
experimental autoimmune encephalomyelitis. Nat Immunol 17, 583-592.
Martin, G.S. (2001). The hunting of the Src. Nat Rev Mol Cell Biol 2, 467-475.
Martinon, F., Agostini, L., Meylan, E., and Tschopp, J. (2004). Identification of bacterial
muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr Biol 14, 1929-
1934.
Marzano, A.V., Damiani, G., Ceccherini, I., Berti, E., Gattorno, M., and Cugno, M. (2017).
Autoinflammation in pyoderma gangrenosum and its syndromic form (pyoderma gangrenosum,
acne and suppurative hidradenitis). Br J Dermatol 176, 1588-1598.
Mason, D.R., Beck, P.L., and Muruve, D.A. (2012). Nucleotide-binding oligomerization domain-
like receptors and inflammasomes in the pathogenesis of non-microbial inflammation and
diseases. J Innate Immun 4, 16-30.
Miller, W.T. (2003). Determinants of substrate recognition in nonreceptor tyrosine kinases.
Accounts of chemical research 36, 393-400.
Mizuno, K., Tagawa, Y., Mitomo, K., Watanabe, N., Katagiri, T., Ogimoto, M., and Yakura, H.
(2002). Src homology region 2 domain-containing phosphatase 1 positively regulates B cell
receptor-induced apoptosis by modulating association of B cell linker protein with Nck and
activation of c-Jun NH2-terminal kinase. J Immunol 169, 778-786.
Moarefi, I., LaFevre-Bernt, M., Sicheri, F., Huse, M., Lee, C.H., Kuriyan, J., and Miller, W.T.
(1997). Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature
385, 650-653.
Modi, B.P., Teves, M.E., Pearson, L.N., Parikh, H.I., Haymond-Thornburg, H., Tucker, J.L.,
Chaemsaithong, P., Gomez-Lopez, N., York, T.P., Romero, R., et al. (2017). Mutations in fetal
genes involved in innate immunity and host defense against microbes increase risk of preterm
premature rupture of membranes (PPROM). Mol Genet Genomic Med 5, 720-729.
Mohapatra, B., Ahmad, G., Nadeau, S., Zutshi, N., An, W., Scheffe, S., Dong, L., Feng, D.,
Goetz, B., Arya, P., et al. (2013). Protein tyrosine kinase regulation by ubiquitination: critical
roles of Cbl-family ubiquitin ligases. Biochim Biophys Acta 1833, 122-139.
178
Moynagh, P.N. (2005). The NF-κB pathway. Journal of Cell Science 118, 4589.
Musumeci, F., Schenone, S., Brullo, C., Desogus, A., Botta, L., and Tintori, C. (2015). Hck
inhibitors as potential therapeutic agents in cancer and HIV infection. Curr Med Chem 22, 1540-
1564.
Naganna, N., Opoku-Temeng, C., Choi, E.Y., Larocque, E., Chang, E.T., Carter-Cooper, B.A.,
Wang, M., Torregrosa-Allen, S.E., Elzey, B.D., Lapidus, R.G., et al. (2019). Amino
alkynylisoquinoline and alkynylnaphthyridine compounds potently inhibit acute myeloid
leukemia proliferation in mice. EBioMedicine 40, 231-239.
Nam, N.H., Pitts, R.L., Sun, G., Sardari, S., Tiemo, A., Xie, M., Yan, B., and Parang, K. (2004).
Design of tetrapeptide ligands as inhibitors of the Src SH2 domain. Bioorg Med Chem 12, 779-
787.
Normand, S., Waldschmitt, N., Neerincx, A., Martinez-Torres, R.J., Chauvin, C., Couturier-
Maillard, A., Boulard, O., Cobret, L., Awad, F., Huot, L., et al. Proteasomal degradation of
NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by
enteropathogens.
Normand, S., Waldschmitt, N., Neerincx, A., Martinez-Torres, R.J., Chauvin, C., Couturier-
Maillard, A., Boulard, O., Cobret, L., Awad, F., Huot, L., et al. (2018). Proteasomal degradation
of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by
enteropathogens. Nature Communications 9, 5338.
Oda, H., Kumar, S., and Howley, P.M. (1999). Regulation of the Src family tyrosine kinase Blk
through E6AP-mediated ubiquitination. Proceedings of the National Academy of Sciences of the
United States of America 96, 9557-9562.
Oeckinghaus, A., and Ghosh, S. (2009). The NF-kappaB family of transcription factors and its
regulation. Cold Spring Harbor perspectives in biology 1, a000034-a000034.
Ogawa, A., Takayama, Y., Sakai, H., Chong, K.T., Takeuchi, S., Nakagawa, A., Nada, S.,
Okada, M., and Tsukihara, T. (2002). Structure of the carboxyl-terminal Src kinase, Csk. J Biol
Chem 277, 14351-14354.
Okada, M. (2012). Regulation of the SRC family kinases by Csk. Int J Biol Sci 8, 1385-1397.
Parameswaran, N., and Patial, S. (2010). Tumor necrosis factor-α signaling in macrophages.
Critical reviews in eukaryotic gene expression 20, 87-103.
Patel, R.K., Weir, M., Hellwig, S., Dorman, H., and Smithgall, T.E. (2017). Abstract 2363:
Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD⁺
AML. Cancer Research 77, 2363-2363.
179
Pecquet, C., Nyga, R., Penard-Lacronique, V., Smithgall, T.E., Murakami, H., Regnier, A.,
Lassoued, K., and Gouilleux, F. (2007). The Src tyrosine kinase Hck is required for Tel-Abl- but
not for Tel-Jak2-induced cell transformation. Oncogene 26, 1577-1585.
Pellicena, P., Stowell, K.R., and Miller, W.T. (1998). Enhanced phosphorylation of Src family
kinase substrates containing SH2 domain binding sites. J Biol Chem 273, 15325-15328.
Pene-Dumitrescu, T., Peterson, L.F., Donato, N.J., and Smithgall, T.E. (2008). An inhibitor-
resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of
the broad-spectrum Src family kinase inhibitor A-419259. Oncogene 27, 7055-7069.
Pene-Dumitrescu, T., and Smithgall, T.E. (2010). Expression of a Src family kinase in chronic
myelogenous leukemia cells induces resistance to imatinib in a kinase-dependent manner. J Biol
Chem 285, 21446-21457.
Pinheiro, A.S., Eibl, C., Ekman-Vural, Z., Schwarzenbacher, R., and Peti, W. (2011). The
NLRP12 pyrin domain: structure, dynamics, and functional insights. Journal of molecular
biology 413, 790-803.
Poh, A.R., Love, C.G., Masson, F., Preaudet, A., Tsui, C., Whitehead, L., Monard, S., Khakham,
Y., Burstroem, L., Lessene, G., et al. (2017). Inhibition of Hematopoietic Cell Kinase Activity
Suppresses Myeloid Cell-Mediated Colon Cancer Progression. Cancer Cell 31, 563-575.e565.
Poh, A.R., O'Donoghue, R.J., and Ernst, M. (2015). Hematopoietic cell kinase (HCK) as a
therapeutic target in immune and cancer cells. Oncotarget 6, 15752-15771.
Poincloux, R., Cougoule, C., Daubon, T., Maridonneau-Parini, I., and Le Cabec, V. (2007).
Tyrosine-phosphorylated STAT5 accumulates on podosomes in Hck-transformed fibroblasts and
chronic myeloid leukemia cells. J Cell Physiol 213, 212-220.
Prada-Arismendy, J., Arroyave, J.C., and Rothlisberger, S. (2017). Molecular biomarkers in
acute myeloid leukemia. Blood Rev 31, 63-76.
Prochnicki, T., and Latz, E. (2017). Inflammasomes on the Crossroads of Innate Immune
Recognition and Metabolic Control. Cell Metab 26, 71-93.
Quintas-Cardama, A., and Cortes, J. (2009). Molecular biology of bcr-abl1-positive chronic
myeloid leukemia. Blood 113, 1619-1630.
Quintrell, N., Lebo, R., Varmus, H., Bishop, J.M., Pettenati, M.J., Le Beau, M.M., Diaz, M.O.,
and Rowley, J.D. (1987). Identification of a human gene (HCK) that encodes a protein-tyrosine
kinase and is expressed in hemopoietic cells. Mol Cell Biol 7, 2267-2275.
Rhodes, D.R., Yu, J., Shanker, K., Deshpande, N., Varambally, R., Ghosh, D., Barrette, T.,
Pandey, A., and Chinnaiyan, A.M. (2004). ONCOMINE: a cancer microarray database and
integrated data-mining platform. Neoplasia 6, 1-6.
180
Rhyasen, G.W., and Starczynowski, D.T. (2014). IRAK signalling in cancer. British Journal Of
Cancer 112, 232.
Robbins, S.M., Quintrell, N.A., and Bishop, J.M. (1995). Myristoylation and differential
palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and
association with caveolae. Mol Cell Biol 15, 3507-3515.
Romanova, L.Y., and Mushinski, J.F. (2011). Central role of paxillin phosphorylation in
regulation of LFA-1 integrins activity and lymphocyte migration. Cell Adh Migr 5, 457-462.
Roversi, F.M., Pericole, F.V., Duarte, A.d.S.S., Ferro, K.P., Corrocher, F.A., Palodetto, B.,
Longhini, A.L., Botta, M., and Saad, S.T.O. (2015). Knockdown of HCK Reduces Cell Death
and Erythroid Differentiation in Human CD34⁺ Hematopoietic Progenitor Cells.
Blood 126, 2860.
Roversi, F.M., Pericole, F.V., Machado-Neto, J.A., da Silva Santos Duarte, A., Longhini, A.L.,
Corrocher, F.A., Palodetto, B., Ferro, K.P., Rosa, R.G., Baratti, M.O., et al. (2017).
Hematopoietic cell kinase (HCK) is a potential therapeutic target for dysplastic and leukemic
cells due to integration of erythropoietin/PI3K pathway and regulation of erythropoiesis: HCK in
erythropoietin/PI3K pathway. Biochim Biophys Acta Mol Basis Dis 1863, 450-461.
Roversi, F.M., Pericole, F.V., Machado-Neto, J.A., Longhini, A.L., Duarte, A.S.S., Palodetto, B.,
Corrocher, F.A., Traina, F., and Olalla Saad, S.T. (2014). Hematopoietic Cell Kinase (HCK) Is
Involved in Myelodysplastic Syndromes and Acute Leukemia Pathophysiology By Modulating
PI3K Pathway and Enhancing Erythroid Differentiation. Blood 124, 1891-1891.
Ryu, S.W., Lee, S.J., Park, M.Y., Jun, J.I., Jung, Y.K., and Kim, E. (2003). Fas-associated factor
1, FAF1, is a member of Fas death-inducing signaling complex. J Biol Chem 278, 24003-24010.
Safley, S.A., Villinger, F., Jackson, E.H., Tucker-Burden, C., Cohen, C., and Weber, C.J. (2004).
Interleukin-6 production and secretion by human parathyroids. Clinical and experimental
immunology 136, 145-156.
Saito, Y., Yuki, H., Kuratani, M., Hashizume, Y., Takagi, S., Honma, T., Tanaka, A., Shirouzu,
M., Mikuni, J., Handa, N., et al. (2013). A pyrrolo-pyrimidine derivative targets human primary
AML stem cells in vivo. Sci Transl Med 5, 181ra152.
Sarvestani, S.T., and McAuley, J.L. (2017). The role of the NLRP3 inflammasome in regulation
of antiviral responses to influenza A virus infection. Antiviral Res 148, 32-42.
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch,
S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for
biological-image analysis. Nat Methods 9, 676-682.
181
Sharif, H., Wang, L., Wang, W.L., Magupalli, V.G., Andreeva, L., Qiao, Q., Hauenstein, A.V.,
Wu, Z., Núñez, G., Mao, Y., et al. (2019). Structural mechanism for NEK7-licensed activation of
NLRP3 inflammasome. Nature 570, 338-343.
Sherry, S.T., Ward, M.H., Kholodov, M., Baker, J., Phan, L., Smigielski, E.M., and Sirotkin, K.
(2001). dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29, 308-311.
Shi, C., Miley, J., Nottingham, A., Morooka, T., Prosdocimo, D.A., and Simon, D.I. (2019).
Leukocyte integrin signaling regulates FOXP1 gene expression via FOXP1-IT1 long non-coding
RNA-mediated IRAK1 pathway. Biochim Biophys Acta Gene Regul Mech 1862, 493-508.
Sicheri, F., Moarefi, I., and Kuriyan, J. (1997). Crystal structure of the Src family tyrosine kinase
Hck. Nature 385, 602-609.
Silveira, T.N., Gomes, M.T., Oliveira, L.S., Campos, P.C., Machado, G.G., and Oliveira, S.C.
(2017). NLRP12 negatively regulates proinflammatory cytokine production and host defense
against Brucella abortus. Eur J Immunol 47, 51-59.
Sims, J.E., and Smith, D.E. (2010). The IL-1 family: regulators of immunity. Nature Reviews
Immunology 10, 89.
Smolinska, M.J., Page, T.H., Urbaniak, A.M., Mutch, B.E., and Horwood, N.J. (2011). Hck
Tyrosine Kinase Regulates TLR4-Induced TNF and IL-6 Production via AP-1. The Journal of
Immunology 187, 6043.
Stewart, R.A., Li, D.M., Huang, H., and Xu, T. (2003). A genetic screen for modifiers of the lats
tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in
Drosophila. Oncogene 22, 6436-6444.
Strauss, J.F., 3rd, Romero, R., Gomez-Lopez, N., Haymond-Thornburg, H., Modi, B.P., Teves,
M.E., Pearson, L.N., York, T.P., and Schenkein, H.A. (2018). Spontaneous preterm birth:
advances toward the discovery of genetic predisposition. Am J Obstet Gynecol 218, 294-
314.e292.
Sun, C., Murata, Y., Imada, S., Konno, T., Kotani, T., Saito, Y., Yamada, H., and Matozaki, T.
(2018). Role of Csk in intestinal epithelial barrier function and protection against colitis.
Biochem Biophys Res Commun 504, 109-114.
Sun, S.-C. (2017). The non-canonical NF-κB pathway in immunity and inflammation. Nature
reviews Immunology 17, 545-558.
Sun, Z., Lv, J., Zhu, Y., Song, D., Zhu, B., and Miao, C. (2015). Desflurane preconditioning
protects human umbilical vein endothelial cells against anoxia/reoxygenation by upregulating
NLRP12 and inhibiting non-canonical nuclear factor-kappaB signaling. Int J Mol Med 36, 1327-
1334.
182
Takeuchi, O., and Akira, S. (2010). Pattern recognition receptors and inflammation. Cell 140,
805-820.
Thaiss, C.A., and Elinav, E. (2015). NF-kappaB Regulation by NLRs: T Cells Join the Club.
Immunity 42, 595-597.
The Cancer Genome Atlas Research Network. (2013). Genomic and Epigenomic Landscapes of
Adult De Novo Acute Myeloid Leukemia. New England Journal of Medicine 368, 2059-2074.
The UniPort Consortium. (2019). UniProt: a worldwide hub of protein knowledge. Nucleic
Acids Res 47, D506-d515.
Tinti, M., Nardozza, A.P., Ferrari, E., Sacco, F., Corallino, S., Castagnoli, L., and Cesareni, G.
(2012). The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays.
N Biotechnol 29, 571-577.
Truax, A.D., Chen, L., Tam, J.W., Cheng, N., Guo, H., Koblansky, A.A., Chou, W.C., Wilson,
J.E., Brickey, W.J., Petrucelli, A., et al. (2018). The Inhibitory Innate Immune Sensor NLRP12
Maintains a Threshold against Obesity by Regulating Gut Microbiota Homeostasis. Cell Host
Microbe 24, 364-378.e366.
Tuncer, S., Fiorillo, M.T., and Sorrentino, R. (2014). The multifaceted nature of NLRP12. J
Leukoc Biol 96, 991-1000.
Turner, C.E. (2000). Paxillin interactions. J Cell Sci 113 Pt 23, 4139-4140.
van Schouwenburg, P.A., Davenport, E.E., Kienzler, A.K., Marwah, I., Wright, B., Lucas, M.,
Malinauskas, T., Martin, H.C., Lockstone, H.E., Cazier, J.B., et al. (2015). Application of whole
genome and RNA sequencing to investigate the genomic landscape of common variable
immunodeficiency disorders. Clin Immunol 160, 301-314.
Vladimer, G.I., Marty-Roix, R., Ghosh, S., Weng, D., and Lien, E. (2013). Inflammasomes and
host defenses against bacterial infections. Curr Opin Microbiol 16, 23-31.
Vladimer, G.I., Weng, D., Paquette, S.W.M., Vanaja, S.K., Rathinam, V.A., Aune, M.H.,
Conlon, J.E., Burbage, J.J., Proulx, M.K., and Liu, Q. (2012). The NLRP12 inflammasome
recognizes Yersinia pestis. Immunity 37, 96-107.
Vogel, C., and Marcotte, E.M. (2012). Insights into the regulation of protein abundance from
proteomic and transcriptomic analyses. Nat Rev Genet 13, 227-232.
Wang, L., Manji, G.A., Grenier, J.M., Al-Garawi, A., Merriam, S., Lora, J.M., Geddes, B.J.,
Briskin, M., DiStefano, P.S., and Bertin, J. (2002). PYPAF7, a novel PYRIN-containing Apaf1-
like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine
processing. J Biol Chem 277, 29874-29880.
183
Wang, P., Zhu, S., Yang, L., Cui, S., Pan, W., Jackson, R., Zheng, Y., Rongvaux, A., Sun, Q.,
Yang, G., et al. (2015). Nlrp6 regulates intestinal antiviral innate immunity. Science 350, 826-
830.
Wang, Q., Liu, F., Zhang, M., Zhou, P., Xu, C., Li, Y., Bian, L., Liu, Y., Yao, Y., Wang, F., et
al. (2018). NLRP12 Promotes Mouse Neutrophil Differentiation through Regulation of Non-
canonical NF-kappaB and MAPK(ERK1/2) Signaling. Int J Biol Sci 14, 147-155.
Warmuth, M., Bergmann, M., Priess, A., Hauslmann, K., Emmerich, B., and Hallek, M. (1997).
The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and
phosphorylates the Grb2-binding site of Bcr. J Biol Chem 272, 33260-33270.
Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de
Beer, T.A.P., Rempfer, C., Bordoli, L., et al. (2018). SWISS-MODEL: homology modelling of
protein structures and complexes. Nucleic Acids Res 46, W296-w303.
Williams, K.L., Lich, J.D., Duncan, J.A., Reed, W., Rallabhandi, P., Moore, C., Kurtz, S.,
Coffield, V.M., Accavitti-Loper, M.A., Su, L., et al. (2005). The CATERPILLER protein
monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and
Mycobacterium tuberculosis-induced pro-inflammatory signals. J Biol Chem 280, 39914-39924.
Williams, K.L., Taxman, D.J., Linhoff, M.W., Reed, W., and Ting, J.P. (2003). Cutting edge:
Monarch-1: a pyrin/nucleotide-binding domain/leucine-rich repeat protein that controls classical
and nonclassical MHC class I genes. J Immunol 170, 5354-5358.
Wu, Y., Shi, T., and Li, J. (2019). NLRC5: A paradigm for NLRs in immunological and
inflammatory reaction. Cancer Lett 451, 92-99.
Xia, X., Dai, C., Zhu, X., Liao, Q., Luo, X., Fu, Y., and Wang, L. (2016). Identification of a
Novel NLRP12 Nonsense Mutation (Trp408X) in the Extremely Rare Disease FCAS by Exome
Sequencing. PLoS One 11, e0156981.
Yarza, R., Vela, S., Solas, M., and Ramirez, M.J. (2015). c-Jun N-terminal Kinase (JNK)
Signaling as a Therapeutic Target for Alzheimer's Disease. Front Pharmacol 6, 321.
Ye, Z., Lich, J.D., Moore, C.B., Duncan, J.A., Williams, K.L., and Ting, J.P. (2008). ATP
binding by monarch-1/NLRP12 is critical for its inhibitory function. Mol Cell Biol 28, 1841-
1850.
Zaki, M.H., Vogel, P., Malireddi, R.K., Body-Malapel, M., Anand, P.K., Bertin, J., Green, D.R.,
Lamkanfi, M., and Kanneganti, T.D. (2011). The NOD-like receptor NLRP12 attenuates colon
inflammation and tumorigenesis. Cancer Cell 20, 649-660.
Zhou, J., Ching, Y.Q., and Chng, W.J. (2015a). Aberrant nuclear factor-kappa B activity in acute
myeloid leukemia: from molecular pathogenesis to therapeutic target. Oncotarget 6, 5490-5500.
184
Zhou, J., Zhang, X., Wang, Y., and Guan, Y. (2015b). PU.1 affects proliferation of the human
acute myeloid leukemia U937 cell line by directly regulating MEIS1. Oncol Lett 10, 1912-1918.
Zhu, F., Choi, B.Y., Ma, W.-Y., Zhao, Z., Zhang, Y., Cho, Y.Y., Choi, H.S., Imamoto, A., Bode,
A.M., and Dong, Z. (2006). COOH-terminal Src kinase-mediated c-Jun phosphorylation
promotes c-Jun degradation and inhibits cell transformation. Cancer research 66, 5729-5736.
Ziegler, S.F., Marth, J.D., Lewis, D.B., and Perlmutter, R.M. (1987). Novel protein-tyrosine
kinase gene (hck) preferentially expressed in cells of hematopoietic origin. Mol Cell Biol 7,
2276-2285.

Abstract (if available)

Abstract The members of the nucleotide-binding oligomerization domain-like receptor (NLR) proteins family are composed of a N-terminal domain, a central nucleotide-binding domain (NBD), and a C-terminal ligand-binding domain. They are generally known to be involved in regulation of the NF-kB signaling pathway. A yeast two-hybrid screen was performed with the cDNA of the pyrin plus nucleotide binding domain (PYD + NBD) of NLR family pyrin domain-containing12 protein (Nlrp12) as bait and a human leucocyte cDNA library as prey. Hematopoiesis cell kinase (Hck), a Src non-receptor tyrosine kinase family member was the top hit. Further experiments confirmed that: 1) the C-terminal 42 amino acids of Hck specifically bound to Nlrp12’s PYD + NBD, but not to the Nlrp 3 and Nlrp 8, and Nlrp12 PYD + NBD domains

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Maternal embryonic leucine zipper kinase (MELK) as a novel therapeutic target in the treatment of acute lymphoblastic leukemia

PDF

Investigation of the synergistic effect of midostaurin, a tyrosine kinase inhibitor, with anti FLT3 antibodies-based therapies for acute myeloid leukemia

PDF

Using cyclotides as a bioscaffold to target intracellular protein-protein interactions

PDF

Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles

PDF

Enhancing the specificity and cytotoxicity of chimeric antigen receptor Natural Killer cells

PDF

Characterization of three novel variants of the MAVS adaptor

PDF

The modulation of dynamin and receptor endocytosis machinery using elastin-like polypeptides

Asset Metadata

Creator Zhang, Yue (author)

Core Title Characterization of the interaction of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 12 (Nlrp12) with hematopoietic cell kinase (Hck)

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 12/12/2019

Defense Date 05/29/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag acute myeloid leukemia,functional studies,Hck,innate immunity,Nlrp12,OAI-PMH Harvest,protein-protein interaction,yeast two-hybrid

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Okamoto, Curtis Toshio (committee chair), Alachkar, Houda (committee member), Xie, Jianming (committee member)

Creator Email zhan576@usc.edu,zhangyue08@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-252926

Unique identifier UC11673994

Identifier etd-ZhangYue-8027.pdf (filename),usctheses-c89-252926 (legacy record id)

Legacy Identifier etd-ZhangYue-8027.pdf

Dmrecord 252926

Document Type Dissertation

Rights Zhang, Yue

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA