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The molecular mechanism underlying the autoimmune-associated PTPN22 R620W variation and the quest for therapeutics

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The molecular mechanism underlying the autoimmune-associated PTPN22 R620W variation and the quest for therapeutics

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Content i
THE MOLECULAR MECHANISM UNDERLYING THE AUTOIMMUNE-ASSOCIATED
PTPN22 R620W VARIATION AND THE QUEST FOR THERAPEUTICS

by

Stephanie Michele Stanford

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)

August 2010

Copyright 2010 Stephanie Michele Stanford

ii
Dedication
This is for Michael, without whose support it would not have happened.
iii
Acknowledgements
I would like to thank my mentor Dr. Nunzio Bottini for his tremendous patience and effort
in training me, but most of all for introducing me to the world of TCR signaling.

Chapter 2: Edoardo Fiorillo, Valeria Orrú, Yingge Liu, Mogjiborahman Salek, Novella
Rapini, Aaron D. Schenone, Patrizia Saccucci, Lucia G. Delogu, Federica Angelini,
Maria Luisa Manca Bitti, Christian Schmedt, Andrew C. Chan, Oreste Acuto

Chapter 3: Divya Krishnamurthy, Roza Kazemi, Bikash Debnath, Matthew D. Falk,
Russell Dahl, Lei Zhao, Kiminori Hasegawa, Andrew C. Chan, Amy M. Barrios, Nouri
Neamati

Chapter 4: Rekha G. Panchal, Sayantan Mitra, Logan M. Walker, Sagar Damle, David
Ruble, Teodora Kaltcheva, Sina Bavari, Amy M. Barrios

I am also grateful to Tomas Mustelin and Torkel Vang for sharing plasmids, to Amy
Barrios and her laboratory (Sayantan Mitra and Teodora Kaltcheva) for providing the
pCAP and CAP peptides, to James MacMurray for critical review of the data, and to
Krishna Kota and Robert C Boltz for high-content image analysis.

I would also like to thank the NIH Training Grant in Cellular, Biochemical, and Molecular
biology at USC for my support.

iv
Table of Contents
Dedication ii

Acknowledgements iii

List of Tables vi

List of Figures vii

Abstract viii

Chapter 1: Lymphoid tyrosine phosphatase and autoimmunity: human genetics
rediscovers tyrosine phosphatases 1
1.1 Abstract
1.2 Tyrosine phosphatases: from TCR signaling to human autoimmunity 1
1.3 LYP is a critical negative regulator of TCR signaling 3
1.4 Association of PTPN22 with human autoimmunity 7
1.5 Functional genetics of the PTPN22 C1858T polymorphism 12
1.6 (Putative) mechanisms of action of PTPN22 in autoimmunity 15
1.7 LYP as a possible drug target for autoimmunity 17

Chapter 2: Autoimmune-associated variation reduces phosphorylation of lymphoid
phosphatase on an inhibitory tyrosine residue 20
2.1 Abstract 20
2.2 Introduction 20
2.3 Experimental Procedures 22
2.4 Results 30
2.5 Discussion 50

Chapter 3: Non-phospho-mimetic inhibitors of lymphoid tyrosine phosphatase 55
3.1 Abstract 55
3.2 Introduction 55
3.3 Experimental Procedures 57
3.4 Results 62
3.5 Discussion 81

Chapter 4: Single-cell assay of tyrosine phosphatase activity using fluorogenic
pCAP peptides 84
4.1 Abstract 84
4.2 Introduction 84
4.3 Experimental Procedures 87
4.4 Results & Discussion 94

Chapter 5: Conclusions 110
5.1 LYP as a drug target for treatment of autoimmunity 11-
5.2 The regulation of LYP in T cells 111
5.3 New assays for PTPs 112

Bibliography 115
v
List of Tables
Table 3.1. Structures of top LYP small molecule inhibitors 64

Table 3.2. Potency and selectivity of top LYP small molecule inhibitors 65

Table 3.3. Compound 4 causes increased T cell activation 72

Table 3.4. Potency and selectivity of substitutions of Domain 1 76

Table 3.5. Potency and selectivity of substitutions of Domain 2 77

Table 3.6. Potency and selectivity of substitutions of Domain 3 80

vi
List of Figures
Figure 2.1. Binding of Csk to LYP does not directly affect the phosphatase activity 32

Figure 2.2. TCR-induced tyrosine phosphorylation of LYP 34

Figure 2.3. LYP-W620 is less phosphorylated in resting and TCR-stimulated T cells 36

Figure 2.4. Phosphorylation of LYP in T cells depends on Csk and Lck 38

Figure 2.5. Csk-dependent co-precipitation of Lck with LYP 41

Figure 2.6. Tyr536 is a major Lck phosphorylation site of LYP 45

Figure 2.7. Tyr536 is a direct phosphorylation site for Lck but not for Csk 46

Figure 2.8. Reduced phosphorylation of Tyr536 on LYP-W620 leads to
gain-of-function inhibition of TCR signaling 48

Figure 2.9. Model of regulation of LYP and Lck activity by reciprocal negative
feedback 54

Figure 3.1. Intracellular inhibition of LYP by compound 4 67

Figure 3.2. Treatment of T cells with compound 4 increases TCR signaling
downstream Lck 70

Figure 3.3. Mechanism of inhibition of compound 4 73

Figure 3.4. Domains of compound 4 75

Figure 4.1. pCAP peptides are specific PTP activity probes 95

Figure 4.2. Detection of intracellular PTP activity in microinjected
cells 96

Figure 4.3. pCAP peptides can be used to monitor intracellular PTP activity 98

Figure 4.4. Optimization of CAP peptide nucleofection into Jurkat
cells 99

Figure 4.5. Fluorescence of non-nucleofected cells incubated in
thepresence of the CAP-based peptides used in Fig. 4.2A 100

Figure 4.6. Optimization of cell-permeable pCAP peptides 102

Figure 4.7. Cytosolic distribution of C14-R7-CAP peptide 102

Figure 4.8. Automated imaging of RAW cells 103
vii

Figure 4.9. Internalization of cell-permeable fluorogenic PTP activity probes 105

Figure 4.10. HCI assay of intracellular PTP activity using cell-permeable pCAP
peptides 107

Figure 4.11. Flow cytometry assay of intracellular PTP activity
using cell-permeable pCAP 108

Figure 4.12. Single-cell assay of YopH activity 108

viii
Abstract
PTPs involved in modulation of signal transduction through the T cell receptor (TCR) are
promising targets for human autoimmunity. Here we will focus on the lymphoid tyrosine
phosphatase LYP, a critical negative modulator of TCR signaling encoded by the
PTPN22 gene. A missense C1858T single nucleotide polymorphism in the PTPN22
gene recently emerged as a major risk factor for multiple human autoimmune diseases.
In T cells, LYP forms a complex with the negative regulatory kinase Csk and is a critical
negative regulator of signaling through the T cell receptor. The C1858T SNP results in
the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits
reduced interaction with Csk and is a gain-of-function inhibitor of TCR signaling. While
strong genetic and functional evidence has suggested that LYP is a promising candidate
drug target for treatment of human autoimmunity, the molecular mechanism of the
autoimmune-associated R620W variation remains unknown.

We hypothesize that an inhibitor of LYP could potentially revert the gain-of-function
effect and restore normal TCR signaling levels in carriers of LYP-W620. While a small
molecule inhibitor would be useful for both understanding the role of LYP in the immune
system and for validating LYP as a drug target, the development of specific, cell-
permeable PTP inhibitors is currently hampered by the lack of methods for high-
throughput screening (HTS) to identify cell-permeable leads.

Here we present the mechanism by which the pathogenic R620W polymorphism of LYP
causes a gain-of-function form of the phosphatase. We also propose two approaches to
identify candidate cell-permeable inhibitors of PTPs. We show that through a novel
screen of a library of drug-like small-molecule compounds, we were able to identify a
ix
non-phospho-mimetic cell-permeable inhibitor of LYP which increases TCR signaling
and T cell activation. We also propose the first cell-based assay to directly monitor PTP
activity at the single-cell level. Our assay can be adapted to screening of inhibitor
libraries for any PTP of interest to identify cell-permeable inhibitors.

1
Chapter 1: Lymphoid tyrosine phosphatase and autoimmunity:
human genetics rediscovers tyrosine phosphatases

The following text has been published as Stanford, S.M., Mustelin, T.M., and Bottini, N.
Lymphoid tyrosine phosphatase and autoimmunity: human genetics rediscovers tyrosine
phosphatases. 2010. Seminars in Immunopathology. This text is reproduced with
permission from Springer.

1.1 Abstract
A relatively large number of protein tyrosine phosphatases (PTPs) are known to regulate
signaling through the T cell receptor (TCR). Recent human genetics studies have shown
that several of these PTPs are encoded by major autoimmunity genes. Here we will
focus on the lymphoid tyrosine phosphatase LYP, a critical negative modulator of TCR
signaling encoded by the PTPN22 gene. The functional analysis of autoimmune-
associated PTPN22 genetic variants suggests that genetic variability of TCR signal
transduction contributes to the pathogenesis of autoimmunity in humans.

1.2 Tyrosine phosphatases: from TCR signaling to human autoimmunity
A relatively large number of protein tyrosine phosphatases (PTPs) are able to either
upregulate or downregulate signaling through the T cell receptor (TCR). For the current
classification of PTPs and an overview of all the PTPs involved in T cell activation, we
refer the reader to the following reviews(Mustelin and Tasken 2003; Alonso, Sasin et al.
2004; Mustelin, Alonso et al. 2004). There is well-established evidence that alteration of
the expression/activity of PTPs involved in TCR signaling regulation causes
immunopathology in mice. One of the first examples reported was the SH2-containing
2
PTP SHP-1, which is encoded by the gene Ptpn6, and is a negative regulator of
signaling through the TCR and other immune receptors. Functional inactivation of SHP-1
in mice gives rise to the motheaten phenotype, characterized by immune-deficiency,
autoimmunity and lymphoproliferation(Kozlowski, Mlinaric-Rascan et al. 1993; Tsui and
Tsui 1994). Another well-known example is the receptor PTP CD45, encoded by the
Ptprc gene, which is a positive regulator of TCR signaling. CD45 is currently considered
a drug target for inflammatory diseases and autoimmunity, based upon several lines of
in vivo evidence, including the fact that CD45 knockout (KO)

mice

show severe
immunodeficiency(Koretzky, Picus et al. 1991), while mice carrying a gain-of-function
E613R mutation develop autoimmune-like disease(Majeti, Xu et al. 2000).

Studies on PTPs in human immunopathology have somehow lagged behind until
recently, when genetic studies have shown that PTPs make up a significant fraction of
the autoimmunity genes in humans. Initial studies using the candidate-gene approach
and more recent genome-wide association (GWA) studies have shown that at least six
major (i.e. showing genome-wide signal in association studies) autoimmunity genes
encode known PTPs (PTPN22, PTPN2, PTPRC, UBASH3A, PTPN11, PTPRT)(Bottini,
Musumeci et al. 2004; 2007; Todd, Walker et al. 2007; Concannon, Onengut-Gumuscu
et al. 2008; Hennig, Fry et al. 2008; Julia, Ballina et al. 2008; Raychaudhuri, Thomson et
al. 2009). Most of these enzymes are known regulators of T cell activation(Carpino,
Turner et al. 2004; Mustelin, Alonso et al. 2004). Among the PTPs encoded by
autoimmunity-genes, only CD45 (encoded by PTPRC) has an established role in mouse
autoimmunity, and excellent reviews have been published on the biochemistry and
immunology of this enzyme(Hermiston, Xu et al. 2003; Irie-Sasaki, Sasaki et al. 2003).
Little is known about the other PTPs and/or their mechanism of action in human
3
autoimmunity. In this review we will focus on the PTP encoded by PTPN22, which is
known as lymphoid tyrosine phosphatase (LYP) and is a potent modulator of TCR
signaling. The results of functional genetics studies of autoimmune-predisposing
PTPN22 variants support the idea that genetic variability of TCR signaling predisposes a
subset of individuals to development of autoimmune diseases. These same studies also
suggest that inhibition of LYP might be beneficial in autoimmunity. If confirmed by further
validation data, this will make LYP one of the first cases of a novel drug target emerging
from human genetic studies.

1.3 LYP is a critical negative regulator of TCR signaling
LYP is a Class 1 cytosolic PTP(Alonso, Sasin et al. 2004), with expression restricted to
hematopoietic cells. It belongs to a sub-family of “PEST-enriched PTPs” (corresponding
to the NT4 subtype of Classical PTPs according to another classification
system(Andersen, Jansen et al. 2004)), which includes two additional enzymes, PTP-
PEST (encoded by the PTPN12 gene), and BDP1 (encoded by the PTPN18 gene).

LYP and its mouse homolog pest-enriched phosphatase (Pep) are ~105 kD proteins
characterized by a ~300 aa N-terminal tyrosine phosphatase domain and a ~200 aa C-
terminal domain which includes four putative polyproline (PEST-enriched) motifs (termed
P1-P4)(Matthews, Bowne et al. 1992; Cohen, Dadi et al. 1999). The catalytic domain
and the C-terminal domain are separated by a ~300 aa region called “the interdomain.”
A second shorter isoform of LYP called LYP2 has been identified in resting T
cells(Cohen, Dadi et al. 1999). Little is known about the expression/function of LYP2 or
the possible existence of additional isoforms of LYP or Pep. However, by western
blotting of lysates of resting or TCR-stimulated human T cells, we observed that full-
4
length LYP is by far the predominant isoform in these cells and that LYP2 expression
levels are much lower than full-length LYP both at the mRNA and protein levels (our
unpublished observation).

In T cells, LYP and Pep are potent negative regulators of TCR signaling(Cloutier and
Veillette 1996; Cloutier and Veillette 1999; Gjorloff-Wingren, Saxena et al. 1999; Hill,
Zozulya et al. 2002; Begovich, Carlton et al. 2004; Vang, Congia et al. 2005) through
dephosphorylation of several key mediators of early TCR signal transduction(Cloutier
and Veillette 1999; Wu, Katrekar et al. 2006). The activatory pTyr residues in the
catalytic domain of the Src kinases Lck (Tyr394) and FynT (Tyr417) and of the Syk
family kinase Zap70 (Tyr493) are considered to be physiological substrates of Pep and
LYP. In addition to these kinases, TCRzeta, CD3epsilon, Vav, and Vcp were also
identified in substrate-trapping experiments and are additional putative substrates of the
phosphatase(Wu, Katrekar et al. 2006). Importantly, in T cells, and presumably in other
immune cells, Pep and LYP form a complex with the tyrosine kinase Csk, also a
negative regulator of TCR signaling(Cloutier and Veillette 1996; Cloutier and Veillette
1999; Begovich, Carlton et al. 2004; Bottini, Musumeci et al. 2004). The complex
between the phosphatase and the kinase is dependent on the interaction between the
most N-terminal P1 motif of Pep/LYP and the SH3 domain of Csk(Cloutier and Veillette
1996). The molecular basis of the Pep/Csk interaction has been elucidated by alanine
scanning of the P1 domain(Gregorieff, Cloutier et al. 1998) and by
crystallography(Ghose, Shekhtman et al. 2001). In T cells the high stoichiometry of the
complex, (around 50% Pep co-precipitates with Csk(Cloutier and Veillette 1999)) points
to an important physiological function. Early experiments carried out in mouse cell lines
overexpressing Pep mutants(Cloutier and Veillette 1999) and in Jurkat cells transfected
5
with Pep and/or Csk(Gjorloff-Wingren, Saxena et al. 1999) suggested that the interaction
between Pep and Csk leads to synergistic inhibition of TCR signaling. However, as
discussed below, recent data emerging from the functional characterization of human
genetic variants of LYP suggest that this model might not be universally valid and/or
exhaustive. More work is needed in order to clarify the function of the LYP-Csk complex
in TCR signaling. The recent finding that the phosphatase is excluded from lipid rafts in
mouse T cells(Davidson, Bakinowski et al. 2003) adds complexity to the problem, since
it is hard to reconcile with the high stoichiometry of the Pep-Csk complex and the main
action of the phosphatase on Lck and Zap70. Also there might be additional
physiological interactors of the P1 or other domains of LYP/Pep beside Csk, and
additional proteins might be recruited to the LYP/Csk complex.

The regulation of LYP/Pep is currently an active area of research. Recently the Zhang
group showed that LYP can be phosphorylated on Ser35 by PKC, with consequent
inhibition of the catalytic activity(Yu, Sun et al. 2007). LYP and Pep contain multiple
additional putative sites for Ser/Thr kinases. As mentioned below, we also recently
observed phosphorylation of LYP on Tyr residues, which might contribute to regulate its
activity. Additional interesting aspects of LYP/Pep regulation are emerging from
molecular and structural studies. By studying truncation mutants of the recombinant
phosphatase, we recently demonstrated that the activity of LYP is modulated by an
intramolecular interaction between the proximal interdomain and the catalytic
domain(Liu, Stanford et al. 2009). Crystallization of the catalytic domain of the
phosphatase in our hands also showed that the enzyme might be regulated by a
reversible oxidation mechanism involving a Cys residue(Tsai, Sen et al. 2009).

6
Relatively little is known about the role of LYP/Pep in the immune system in vivo.
Andrew Chan’s group published in early 2004 the phenotype of a global Ptpn22 KO
mouse(Hasegawa, Martin et al. 2004). Deletion of the phosphatase caused expansion of
the T cell memory compartment, and increased TCR signaling in effector T cells. At the
thymic level there was a phenotype of increased positive selection(Hasegawa, Martin et
al. 2004). The phenotype of the mouse supports the idea (originally proposed by Andre
Veillette(Cloutier and Veillette 1996)) that Pep is a key negative regulator of TCR
signaling. The prominent effector/memory T cell phenotype is in line with the high
expression of the phosphatase in these cells ((Zikherman, Hermiston et al. 2009) and
our unpublished observation). Pep is highly expressed in thymocytes as well
((Zikherman, Hermiston et al. 2009) and our unpublished observation), and its role in
thymic selection might help explain the effect of the phosphatase in human
autoimmunity.

An open area of discussion is whether LYP and Pep play any role in regulation of B cell
activation. The KO mouse shows no alterations of signaling through the B cell receptor
(BCR)(Hasegawa, Martin et al. 2004) and low expression levels of Pep have been found
in mouse B cells(Zikherman, Hermiston et al. 2009). Expansion of B cells in germinal
centers occurs in aging mice, but the phenotype has been attributed to increased T cell
help(Hasegawa, Martin et al. 2004). However, a recent study suggested that LYP might
affect signaling through the BCR in primary human B cells ((Rieck, Arechiga et al. 2007)
and see below). Thus it is possible that the KO mouse is a less faithful model of LYP
function in human B cell physiology due to compensation phenomena or perhaps to real
functional differences between the human and the mouse phosphatase. Besides T cells
and B cells, little is known about the role of LYP/Pep in other immune cell
7
subpopulations. For example, a simple search of the BioGPS database(Wu, Orozco et
al. 2009) (http://biogps.gnf.org) suggests that dendritic cells (DC) and natural killer (NK)
cells carry high mRNA levels of LYP/Pep. More immunological studies in the available
KO mouse model are warranted. However, in order to fully understand the role of
LYP/Pep in the immune system, we will also need to generate additional knockout and
transgenic models, engineered in order to enable deletion/overexpression of the
phosphatase in selected immune cell subpopulations.

1.4 Association of PTPN22 with human autoimmunity
Three reports in 2004 documented the association between a missense C1858T
(R620W) single nucleotide polymorphism (SNP) in PTPN22 and type 1 diabetes
(T1D)(Bottini, Musumeci et al. 2004), rheumatoid arthritis (RA)(Begovich, Carlton et al.
2004) and systemic lupus erythematosus (SLE)(Kyogoku, Langefeld et al. 2004). The
association between PTPN22 and T1D, RA and SLE was subsequently widely replicated
by us and others (for example see(Smyth, Cooper et al. 2004; Orozco, Sanchez et al.
2005; Plenge, Padyukov et al. 2005)), and extended to many additional autoimmune
diseases, including Graves’ disease(Smyth, Cooper et al. 2004; Skorka, Bednarczuk et
al. 2005; Heward, Brand et al. 2007), Addison’s disease(Velaga, Wilson et al. 2004;
Skinningsrud, Husebye et al. 2008), vitiligo(Canton, Akhtar et al. 2005; LaBerge, Bennett
et al. 2008), myasthenia gravis(Vandiedonck, Capdevielle et al. 2006; Chuang, Strobel
et al. 2009; Greve, Hoffmann et al. 2009) and systemic sclerosis(Gourh, Tan et al.
2006). PTPN22 *T1858 behaves as a dominant variant, conferring increased risk of
disease already when present in single copy(Bottini, Vang et al. 2006; Gregersen, Lee et
al. 2006). The risk conferred by PTPN22 is variable among diseases, but it is substantial
in T1D and RA, with average reported odds ratios (OR) of 1.7-2.0 per single allele copy.
8
Recent GWA studies detected a major signal on PTPN22 for T1D and RA, and PTPN22
currently ranks in third and second position respectively for single-gene contribution to
the risk of T1D and RA in Caucasian populations(Todd, Walker et al. 2007). In
SLE(Kaufman, Kelly et al. 2006) and T1D(Chelala, Duchatelet et al. 2007), the effect of
PTPN22 seems to be even more prominent in the subgroup of patients who have familial
autoimmunity.

As mentioned, the association of PTPN22 C1858T with autoimmune diseases has been
replicated across different populations. For example, the association with T1D has been
replicated in the American(Ladner, Bottini et al. 2005; Zheng and She 2005),
Italian(Saccucci, Del Duca et al. 2008), German(Kahles, Ramos-Lopez et al. 2005),
Spanish(Santiago, Martinez et al. 2007), English(Smyth, Cooper et al. 2004),
Estonian(Douroudis, Prans et al. 2008), and Ukraininian(Fedetz, Matesanz et al. 2006)
populations. These studies also showed that there are significant differences in the
frequency of the *T1858 allele among populations. The *T1858 allele is more frequent
(around 12.5%) in Northern than in Southern Europeans, while it is almost absent in
African-American and Asian populations(Mori, Yamada et al. 2005; Ikegami, Kawabata
et al. 2007; Zhang, Chen et al. 2008; Lee, Korman et al. 2009). Possible explanations for
such dramatic geographic differences in allele frequency are that the *T1858 allele
appeared recently during evolution and/or its frequency is severely affected by selection.
By analyzing extended haplotype homozygosity, McPartland et al showed evidence of
positive selection at the PTPN22 locus(McPartland, Norris et al. 2007). One intriguing
hypothesis is that the *T1858 allele confers protection toward some highly prevalent
infectious disease. Only a few studies have been carried out on PTPN22 in infectious
diseases so far. Chapman et al found that the *T1858 allele might increase risk of
9
complication in patients affected by common pneumococcal infections(Chapman, Khor
et al. 2006), while another study on immunocompromised transplant recipients found an
opposite protective effect of the *T1858 allele against infections(Azarian, Busson et al.
2008). In tuberculosis (TB) we found some evidence that the *T1858 allele might play a
protective role(Gomez, Anaya et al. 2005; Lamsyah, Rueda et al. 2009). This is an
interesting finding considering that TB might have been a powerful selective force in
Northern Europe. However these data need to be replicated and backed by more
mechanistic studies before any conclusions can be drawn about the possible role of TB
in shaping selection at the PTPN22 locus.

PTPN22 C1858T belongs to a growing family of “shared autoimmunity loci”, which are
associated with multiple autoimmune diseases. Together with a subset of these genes,
PTPN22 also has been shown to contribute to the recurrence of multiple different
autoimmune diseases in certain families(Criswell, Pfeiffer et al. 2005). The identification
and functional dissection of shared autoimmunity loci could potentially unravel major
pathogenic mechanisms of autoimmunity. Association with certain but not other
autoimmune diseases also can give important hints on the mechanism of action of
shared autoimmunity loci. For PTPN22, some of the negative findings might be due to
variable combinations of small effect, low frequency of the allele, and sometime low
statistical power of the study. However, well-powered studies found no association with
multiple sclerosis, and celiac disease(Rueda, Nunez et al. 2005; Zhernakova, Eerligh et
al. 2005; Lee, Rho et al. 2007; Smyth, Plagnol et al. 2008). The *T1858 allele was
reported to have negligible effect on the risk of inflammatory bowel disease (for example
see (van Oene, Wintle et al. 2005) and (Prescott, Fisher et al. 2005)), but a recent large
study suggests that in Crohn’s disease it might be a protective factor(Barrett, Hansoul et
10
al. 2008). A similar protective effect has been reported in Behcet’s disease(Baranathan,
Stanford et al. 2007). Interestingly, PTPN22 *T1858 is associated with psoriatic
arthritis(Butt, Peddle et al. 2006; Huffmeier, Reis et al. 2006) but not with psoriasis.
However, a psoriasis locus might be located in close proximity to PTPN22(Huffmeier,
Steffens et al. 2006; Smith, Warren et al. 2008), suggesting that PTPN22 might lay
within an autoimmunity hot spot on chromosome 1. The reason why PTPN22 associates
with some but not other major autoimmune diseases is unclear at the moment. It has
been suggested that the PTPN22 C1858T polymorphism preferentially associates with
diseases characterized by a strong autoantibody (auto-Ab) component(McGonagle, Aziz
et al. 2009). In favor of this hypothesis, PTPN22 associates with diseases which are
exquisitely auto-Ab-mediated, like myasthenia gravis; also it has been reported by some
groups that the association between PTPN22 and RA is restricted to the anti-cyclic
citrullinated peptide (anti-CCP) Ab positive subgroup(Kallberg, Padyukov et al. 2007).
However, it is unclear whether the association of PTPN22 with auto-Ab is a causal one
and some observations don’t fit well with this theory, for example PTPN22 does not
associate with celiac disease, a disease characterized by auto-Ab production.

Genetic dissection and linkage studies in RA and T1D have determined that PTPN22
C1858T is a primary disease locus(Onengut-Gumuscu, Ewens et al. 2004; Carlton, Hu
et al. 2005; Michou, Lasbleiz et al. 2007; Smyth, Cooper et al. 2008; Zoledziewska,
Perra et al. 2008). Studies are ongoing to clarify whether there are additional
predisposition/protection loci within or close to PTPN22. We reported a rare missense
variation R263Q within the catalytic domain, which is functional (see below) and
segregates on different haplotypes than the R620W variant(Orru, Tsai et al. 2009). A
recent comprehensive analysis of the T1D Genetic Consortium (T1DGC) collection has
11
confirmed the primary association between T1D and C1858T and found evidence for an
independent protective haplotype (Steck et al, manuscript in press). Also there is some
evidence that a variation G-1123C in the promoter region of PTPN22 associates with
autoimmunity(Kawasaki, Awata et al. 2006). The data on this SNP have been difficult to
interpret due to the strict linkage disequilibrium (LD) between the G-1123C and the
C1858T SNPs in European populations(Cinek, Hradsky et al. 2007). In the Sardinian
population, where LD between the two variants is lower, we could detect a strong signal
at the C1858T locus only, suggesting that in the presence of C1858T, the effect of G-
1123C might be a minor one(Zoledziewska, Perra et al. 2008). However, the G-1123C
polymorphism might be a significant risk factor in Asian populations, and more work is
needed in order to establish the association of this SNP with autoimmunity and its
functional effect.

Other important areas of genetic research concern possible interactions between
PTPN22 and other genetic or environmental factors, and whether PTPN22 associates
with prognosis and disease variables other than incidence. Some groups reported that
the risk conferred by PTPN22 depends on HLA genotypes(Steck, Liu et al. 2006), while
no PTPN22-HLA interaction has been found in other studies(Santiago, Martinez et al.
2007; Costenbader, Chang et al. 2008; Morgan, Thomson et al. 2009). Few studies
reported interactions with environmental factors, for example smoking in
RA(Costenbader, Chang et al. 2008; Mahdi, Fisher et al. 2009), and early exposure to
cow milk in T1D(Lempainen, Vaarala et al. 2009). In some populations, the association
between PTPN22 and autoimmunity seems to depend on gender(Kahles, Ramos-Lopez
et al. 2005; Pierer, Kaltenhauser et al. 2006), however this finding has not been
universally confirmed and it is difficult to interpret at the moment. As for the clinical
12
variability of disease, beside the mentioned data on the association with auto-Ab-positive
subsets of RA, there are sporadic reports about a possible effect of PTPN22 on age of
onset(Ladner, Bottini et al. 2005; Santiago, Martinez et al. 2007). The possible value of
PTPN22 in early diagnosis/prognosis has been assessed in prospective studies in T1D
and RA. An association with radiographic progression of disease has been reported in
RA(Lie, Viken et al. 2007). The data in T1D are less homogeneous, and association with
progression to disease has been found in some studies(Hermann, Lipponen et al. 2006)
but not in others(Butty, Campbell et al. 2008), including a notable one, which reported
exclusive association with development of persistent auto-Abs(Steck, Zhang et al. 2009).

1.5 Functional genetics of the PTPN22 C1858T polymorphism
Understanding the effect of the R620W polymorphism on the function of LYP/Pep is
obviously of critical importance. Several functional genetics studies have focused on
possible differences in signal transduction through the TCR in cells from carriers of the
W620 variant of LYP when compared to subjects carrying exclusively the R620 variant.
We reported that primary T cells from T1D patients carrying the W620 allele exhibited
reduced IL-2 response to TCR engagement. These findings were replicated by Aarnisalo
et al in T1D(Aarnisalo, Treszl et al. 2008). The Buckner group reported decreased TCR-
induced phosphorylation of early signaling intermediates, and Ca
++
mobilization in T cells
from *T1858 homozygous RA patients(Rieck, Arechiga et al. 2007). Importantly, the
same group also extended their studies to B cells, and reported that BCR signaling in
primary B cells from carriers of the *T1858 allele is also reduced(Rieck, Arechiga et al.
2007). Thus the vast majority of reports points to decreased TCR-mediated T cell
activation in primary T cells from carriers of the LYP-W620 variant. The only exception
so far is a single report on myasthenia gravis, where the R620W polymorphism
13
associated with increased TCR-induced IL-2 production(Lefvert, Zhao et al. 2008),
however this finding has not been replicated by another group(Chuang, Strobel et al.
2009). Further functional genetics studies on large samples of primary T cells from
healthy controls are ongoing in several laboratories, and hopefully will help solidify even
further our view of the effect of the polymorphism on T cell activation.

Although most studies have focused so far on T cells and on readouts of TCR signaling,
as mentioned, the polymorphism seems to affect signaling through the BCR as
well(Rieck, Arechiga et al. 2007). It is currently unknown whether the polymorphism
affects the function of other cell types which are critical for autoimmunity, for example
DC and NK/NKT cells.

Few studies have looked so far at possible effects of the polymorphism on the
phenotype of primary T and B cells from genotyped patients and controls. For example,
a study suggested that *T1858 carriers have increased numbers of memory T cells, and
that T cells from these individuals might secrete less of the immunosuppressive IL-10
cytokine(Rieck, Arechiga et al. 2007). Much progress is expected in this area in the near
future, which will shed light on the mechanism of action of the polymorphism in
autoimmunity.

At the molecular level, the PTPN22 C1858T polymorphism leads to a substitution of a
Trp for an Arg at position 620. This is a non-conservative variation of a residue within the
P1 motif, which had been previously shown to be critical for the Pep-Csk binding. As a
consequence, the autoimmune-associated LYP-W620 variant exhibits reduced
interaction with Csk(Begovich, Carlton et al. 2004; Bottini, Musumeci et al. 2004).
14
Besides its effect on the complex between the kinase and the phosphatase, the
mechanism of action of the polymorphism at the biochemical and molecular level is still
poorly understood. By studying T cells over-expressing LYP-W620 or LYP-R620, we
found that LYP-W620 expression induces a gain-of-function decrease of TCR-induced
phosphorylation of early key signaling players, and decreased TCR-induced Ca
++

mobilization(Vang, Congia et al. 2005). In our hands also the autoimmune-associated
LYP-W620 variant showed increased phosphatase activity when compared to LYP-
R620(Vang, Congia et al. 2005). We concluded that gain-of-function inhibition of TCR
signaling by LYP-W620, mediated at least in part by increased phosphatase activity, is
responsible for the decreased T cell activation observed in carriers of the pathogenic
phosphatase variant. These conclusions have been recently challenged by a report from
Zikherman et al, claiming that the W620 mutant of LYP (and the homolog W619 mutant
of Pep) is a hypomorph variant, and the gain-of-function phenotype is an artifact which
arises when the phosphatase is overexpressed without parallel overexpression of
Csk(Zikherman, Hermiston et al. 2009). However, it is unclear whether the system used
by Zikherman et al (based on co-transfection of cells with LYP/Pep, Csk, and GFP)
recapitulates more faithfully the physiological stoichiometry of the interaction between
the phosphatase and the kinase. It also remains to be explained how an hypomorph
allele of PTPN22 would give rise to the anomalies of T cell activation reported in primary
T cells from LYP-W620 carriers. More studies in multiple systems including cells from
mice carrying knock-in mutations of LYP/Pep (which are in preparation in several
laboratories in the world) are needed in order to better define the effect of the mutation
on cell signaling.

15
We and others are also actively investigating how the mutation affects the function of
LYP at the molecular level. LYP mRNA levels seem to be unaffected by the
polymorphism, suggesting that the mutation acts mainly at the protein level(Nielsen,
Barington et al. 2007). We recently found that LYP undergoes Csk-dependent
phosphorylation on at least one tyrosine, which results in inhibition of the phosphatase
activity. We suggest that anomalies in Csk-dependent post-translational modification(s)
of LYP mediate at least in part the molecular mechanism of the R620W variation (Fiorillo
et al, manuscript submitted). Csk-independent mechanisms are also possible, including
impaired binding to additional interactors and/or recruitment of LYP-W620 to an
unknown protein through its P1-W620 motif. One of the challenges of the current studies
at the molecular level is to reconcile the gain-of-function phenotype of LYP-W620 with
previous results suggesting a model of synergistic inhibition of signaling by the complex
between the kinase and the phosphatase(Cloutier and Veillette 1999; Gjorloff-Wingren,
Saxena et al. 1999). A possible explanation is that the regulation of LYP and Pep is not
entirely conserved: indeed the two phosphatases share relatively low (<60%) identity in
the interdomain, which might be important for regulation.

1.6 (Putative) mechanisms of action of PTPN22 in autoimmunity
Our current working model is that gain-of-function inhibition of signaling in T cells (and
perhaps other cell types) mediates the pathogenic action of the polymorphism and the
increased risk of autoimmunity of carriers of LYP-W620. It might sound a little counter-
intuitive that decreased immune cell activation leads to autoimmunity, however our
model is in line with the current view that decreased TCR signaling increases risk of at
least a subset of autoimmune diseases. Decreased TCR signaling has been reported in
T cells from NOD mice(Zhang, Salojin et al. 1998) and in peripheral T cells from T1D
16
patients(Buchs and Rapoport 2000). Mechanistic evidence of a link between decreased
TCR signaling and autoimmunity was also provided by the description of the SKG
mouse by Sakaguchi et al, in 2003(Sakaguchi, Takahashi et al. 2003). In this mouse a
loss of function mutation of Zap70 (one of the physiological substrates of LYP) causes a
spontaneous autoimmune disease which is remarkably similar to human RA. Similar
findings of autoimmune-like disease in mice carrying loss-of-function of Zap70 have
been subsequently reported by two additional groups(Siggs, Miosge et al. 2007; Hsu,
Tan et al. 2009). These studies also are showing that decreased TCR signaling
impinges on RA through multiple mechanisms(Siggs, Miosge et al. 2007). In the case of
PTPN22, we could also speculate that some mechanisms might be prominent over
others in different diseases, or even in different subjects affected by the same disease.

One of the favored models is that gain-of-function inhibition of TCR signaling at the
thymic level leads to decreased negative selection and elimination of potentially
autoreactive T cells, and/or decreased production of natural regulatory T cells (Treg).
The observations that 1) Pep is highly expressed in the thymus; 2) the Ptpn22 KO
mouse shows anomalies of thymic selection, and 3) normalization of TCR signaling in
the thymus is able to rescue the phenotype of the SKG mouse(Sakaguchi, Takahashi et
al. 2003) provide additional support to the “thymic selection” theory. In addition or
alternative to thymic selection anomalies, other mechanisms have been envisioned. For
example an anomalous increase in the activity of LYP in effector T cells might negatively
impact the activity/expansion of peripheral Treg in carriers of LYP-W620(Marson,
Kretschmer et al. 2007). Decreased TCR-induced production of IL-2 by effector T cells is
known to impair expansion of Treg in mouse T1D, and neutralization of IL-2 in vivo
induces autoimmunity in mice through a Treg-mediated mechanism(Setoguchi, Hori et
17
al. 2005; Yamanouchi, Rainbow et al. 2007; Tang, Adams et al. 2008). Marson et al also
recently identified PTPN22 among a group of genes that are highly occupied by FoxP3
in Treg. The same study also showed that Treg carry lower levels of the phosphatase
compared to effector T cells(Marson, Kretschmer et al. 2007), suggesting a direct
mechanism by which gain-of-function of LYP can affect Treg function. Since TCR
signaling intensity is an important regulator of naïve T cell differentiation, it is also
possible that decreased TCR signaling affects polarization of naïve T cells into
specialized Thelper populations, for example favoring the appearance of diabetogenic
IFNgamma-producing Th1 cells, in carriers of LYP-W620. The observation that
peripheral T cells from LYP-W620 carriers have a different cytokine secretion pattern
characterized by reduced production of IL-10 is in line with this hypothesis. Other
mechanisms are possible, and, considering the association between PTPN22 and the
presence of auto-Ab, we cannot exclude that LYP-W620 also affects B cell differentiation
and/or tolerization directly.

1.7 LYP as a possible drug target for autoimmunity
Several academic laboratories have began developing small molecule inhibitors of
LYP(Yu, Sun et al. 2007; Xie, Liu et al. 2008; Wu, Bottini et al. 2009). Given the
increased activity of LYP-W620, it has been hypothesized that a selective inhibitor of
LYP could revert the negative effects of the W620 variant on TCR signaling, and thus
constitute an effective etiological therapy of autoimmunity in carriers of the autoimmune-
predisposing variant. Because PTPN22 is implicated in many prevalent autoimmune
diseases (and perhaps even in transplant rejection(Sfar, Gorgi et al. 2009)), an anti-LYP
drug could be of broader value for the treatment of multiple human conditions. Also, the
18
phenotype of the KO mouse suggests that a specific inhibitor of PTPN22 is likely to have
limited side effects.

Although it remains to be proven that the mechanism of action of LYP in autoimmunity is
reversible, the success of trials targeting T1D with monoclonal antibodies against CD3
supports the idea that positive modulation of the TCR helps re-establish tolerance in at
least a subset of autoimmune patients(Chatenoud, Thervet et al. 1994; Chatenoud
2006). In support of the above-mentioned hypothesis, we also recently found a rare loss-
of-function LYP-R263Q genetic variation, which segregates on haplotypes different from
the R620W one, and plays a protective role in SLE(Orru, Tsai et al. 2009).

Further studies in animal models are needed in order to validate LYP as a drug target for
autoimmunity. Importantly, therapeutic inhibition of LYP in autoimmunity might also carry
some risks. For example, excessive inhibition of LYP activity might lead to parallel
counter-productive increase in TCR signaling in effector T cells. Although the Ptpn22 KO
mutation on the B6 background fails to induce autoimmunity(Hasegawa, Martin et al.
2004), Zikherman et al recently showed that crossing of the Ptpn22 KO mouse with the
CD45 E613R (“wedge”) model, led to more severe autoimmunity(Zikherman, Hermiston
et al. 2009). Since spontaneous autoimmunity on B6 background in the CD45 wedge
model is driven by increased TCR(Hermiston, Zikherman et al. 2009) and BCR
signaling(Hermiston, Tan et al. 2005; Gupta, Hermiston et al. 2008), the results of
Zikherman et al suggest that inhibition of LYP might be counterproductive in diseases or
subsets of diseases with a strong component of TCR or BCR hyperactivity (which has
been observed in large subsets of lupus patients(Liossis, Kovacs et al. 1996; Kammer,
19
Perl et al. 2002; Khan, Tsokos et al. 2003)) and thus not easily extended to non-carriers
of gain-of-function LYP mutation(s).

20
Chapter 2: Autoimmune-associated variation reduces phosphorylation of
lymphoid phosphatase on an inhibitory tyrosine residue
This research was originally published in the Journal of Biological Chemistry. Fiorillo et
al. Autoimmune-associated PTPN22 R620W variation reduces phosphorylation of
lymphoid phosphatase on an inhibitory tyrosine residue. The Journal of Biological
Chemistry. 2010; Jun 9. [Epub ahead of print]. © the American Society for Biochemistry
and Molecular Biology(Fiorillo, Orru et al. 2010).

2.1 Abstract
A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently
emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid
tyrosine phosphatase LYP, which forms a complex with the kinase Csk and is a critical
negative regulator of signaling through the T cell receptor. The C1858T SNP results in
the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a
greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here
we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent
manner. TCR-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue
releases tonic inhibition of signaling by LYP. The R620W variation disrupts the
interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which
ultimately contributes to gain-of-function inhibition of T cell signaling.

2.2 Introduction
A C1858T single nucleotide polymorphism [SNP] in PTPN22 was first reported to be
associated with type 1 diabetes [T1D](Bottini, Musumeci et al. 2004) and rheumatoid
arthritis [RA](Begovich, Carlton et al. 2004), and was subsequently found to predispose
21
humans to a wide range of autoimmune diseases(reviewed in (Bottini, Vang et al. 2006;
Gregersen, Lee et al. 2006)). In Caucasian populations PTPN22 currently ranks in third
and in second place in terms of single-gene contribution to the etiology of T1D and RA
respectively (Todd, Walker et al. 2007). PTPN22 *T1858 acts as a dominant allele and
confers significant predisposition to autoimmunity even when present as a single
copy(Bottini, Vang et al. 2006; Gregersen, Lee et al. 2006).

PTPN22 encodes the lymphoid tyrosine phosphatase LYP, which acts as a critical
negative regulator of T cell receptor [TCR] signaling(Cloutier and Veillette 1999; Gjorloff-
Wingren, Saxena et al. 1999; Hill, Zozulya et al. 2002; Begovich, Carlton et al. 2004;
Vang, Congia et al. 2005) through dephosphorylation of several key substrates,
including the Src-family kinases Lck and Fyn, ZAP70 and TCRzeta(Cloutier and Veillette
1999; Wu, Katrekar et al. 2006).

LYP and its mouse homolog PEST-enriched phosphatase [PEP] are ~105 kD proteins
characterized by a ~300 aa N-terminal protein tyrosine phosphatase [PTP] domain and a
~200 aa C-terminal domain which includes four putative polyproline motifs (termed P1-
P4). The catalytic domain and the C-terminal domain are separated by a ~300 aa region
called “the interdomain.” A second shorter isoform of LYP called LYP2 has been
identified in resting T cells(Cohen, Dadi et al. 1999). The most N-terminal P1 motif of
LYP mediates the interaction of PEP/LYP with the SH3 domain of the protein tyrosine
kinase [PTK] Csk, also a negative regulator of TCR signaling(Cloutier and Veillette 1996;
Cloutier and Veillette 1999). The PTPN22 C1858T polymorphism causes an R620W
substitution within the P1 domain of the protein. The pathogenic LYP-W620 variant
exhibits reduced interaction with Csk(Begovich, Carlton et al. 2004; Bottini, Musumeci et
22
al. 2004), shows increased phosphatase activity and is a gain-of-function inhibitor of
signaling in T cells(Vang, Congia et al. 2005; Rieck, Arechiga et al. 2007). T cells from
T1D and healthy subjects carrying the LYP-W620 variant show reduced production of IL-
2 and other cytokines following TCR stimulation(Vang, Congia et al. 2005; Rieck,
Arechiga et al. 2007; Aarnisalo, Treszl et al. 2008). Reduced TCR signaling has recently
been recognized as a major risk factor for autoimmunity, and it affects tolerance through
multiple mechanisms(Sakaguchi, Takahashi et al. 2003; Siggs, Miosge et al. 2007).

Here we sought to identify molecular mechanisms which contribute to the gain-of
function phenotype of LYP-W620 in T cells.

2.3 Experimental Procedures
2.3.1 Plasmids
Full length LYP-R620, LYP-W620, LYP2-R620, and their C227S mutants were cloned in
the BamHI site of the plasmid pEF5-HA(Rahmouni, Vang et al. 2005), while full-length
PEP-R619 and PEP-W619 and their C227S mutants were cloned in the pEFHA vector
(Saito, Williams et al. 2007). Point mutagenesis of LYP constructs was performed by
PCR using primers containing the desired mutation. Flag-tagged LYP-R620 C227S and
amino-terminal truncation mutants of LYP were performed by PCR using LYP-R620 or
LYP-W620 in pEF5-HA ( ∆288LYP) or in pEFHA ( ∆399LYP and ∆517LYP) vector as
templates. Primers were designed in order to anneal around the truncated regions of the
gene and replace the HA tag with a Flag tag. An S-tag (15 aa, see reference(Kim and
Raines 1993)) was cloned 3’ and in frame with the HA tag in the pEF5 vector, thus
generating the pEF5HA-S vector. LYP mutants were then subcloned into the BamHI site.

23
2.3.2 Antibodies and Other Reagents
The anti-HA mAb (clone 16B12) was from Covance (Berkeley, CA). The anti-pTyr Ab
(clone 4G10) was from Chemicon International (Temecula, CA). The anti-LYP pAb was
from R&D Systems (Minneapolis, MN). The anti-PEP pAb has been previously described
(Hasegawa, Martin et al. 2004). The monoclonal anti-Lck, the polyclonal anti-Csk and
the polyclonal anti-Fyn were from Santa Cruz Biotech (Santa Cruz, CA). The anti-Lck
pAb, the monoclonal anti-Fyn, the monoclonal anti-Csk, the anti-huCD4, anti-moCD4
and anti-moCD28 were from BD Biosciences (Carlsbad, CA). The anti-ZAP70 Ab was
from Invitrogen (Carlsbad, CA), while the anti-Itk Ab was from Cell Signaling Technology
(Boston, MA). OKT3 (Kung, Goldstein et al. 1979) was purified from hybridoma
supernatants. F(ab’)
2
Ab and anti-mouse IgG used for cross-linking were purchased from
Jackson Immunoresearch (West Grove, PA) and Upstate/Millipore (Billerica, MA)
respectively. Agarose-conjugated M2-FLAG and PT66 Abs were from Sigma (St. Louis,
MO). PP2 was from EMD Calbiochem (Gibbstown, NJ). The anti-Fyn siRNA was a
commercially available oligo from Santa Cruz, and the anti-Csk and anti-Lck siRNAs
were custom ordered from Dharmacon (Lafayette, CO) (Vang, Abrahamsen et al. 2004;
Methi, Ngai et al. 2005). PfuUltra polymerase was from Stratagene (La Jolla, CA), and
Taq polymerase was from Invitrogen. The AlexaFluor-conjugated anti-HA antibodies
were from Cell Signaling Technology (Boston, MA). The PE-conjugated anti-phospho-
ZAP70(Y319) antibody was from BD Biosciences. The APC-conjugated anti-human
CD69 was purchased from BioLegend (San Diego, CA).

2.3.3 Purification of recombinant full-length LYP
Full-length LYP-R620 and -W620 and their C227S mutants were cloned in the BamHI
site of the pFastBac-HTa (Invitrogen) in frame with a FLAG tag, and recombinant
24
bacmids and baculoviruses were produced using the Bac-to-Bac® method (Invitrogen).
Virus titers/times of incubation were optimized in order to obtain high expression of full-
length recombinant proteins in Sf9 cells. The protein was purified from lysates of insect
cells using single-step affinity chromatography on FLAG-M2 beads, and eluting with a
combination of FLAG peptide and high concentrations of DTT. The final buffer was 50
mM Tris/HCl pH 8.0, 0.5 mM EDTA, and 1 mM DTT. The purity of the recombinant
proteins was more than 80% as assessed by silver stain of polyacrylamide gels (Fig. 1).
The yield of the isolation was around 1 µg full-length protein/150 mm plate of infected
Sf9 cells.

2.3.4 Cell culture, transfection, stimulation
Jurkat E6.1, JTAg (Shaw, Utz et al. 1988), JCaM1, Hut78, and primary T cells were
grown in RPMI 1640 medium supplemented with 10% FCS, 2mM L-glutamine, 1 mM
sodium pyruvate, 10 mM HEPES pH 7.3, 2.5 mg/mL D-glucose, 100 units/mL of
penicillin and 100 µg/mL streptomycin. COS-7 cells were grown in DMEM with 10%
FCS, and 100 units/mL of penicillin and 100 µg/mL streptomycin. Jurkat and JTAg
transfections were performed as described (Vang, Congia et al. 2005). To generate
JTAg cells stably expressing ∆288LYP, cells were transfected with linearized plasmid,
and after 2 days were subjected to selection with 0.2 mg/ml Zeocin (Invitrogen). Stable
transfectants were used for experiments at the polyclonal stage. COS cells were
transfected using Lipofectamine and Plus Reagent (Invitrogen, Carlsbad, CA). For
pervanadate (PV) stimulation of JTAg, a PV solution was added to RPMI to achieve a
200 µM PV final concentration. C305 stimulation of JTAg and Hut78 cells was
performed using C305 hybridoma (Weiss and Stobo 1984) supernatant. For stimulation
of primary human T cells, cells were incubated in RPMI with OKT3 (1 µg/ml) and anti-
25
huCD4 (1 µg/ml) or anti-huCD28 (1 µg/ml), followed by cross-linking with 10 µg/ml anti-
mF(ab’)2 for the time indicated in the figure. For stimulation of mouse thymocytes, cells
were incubated in RPMI with biotinylated anti-moCD3 (20 µg/ml) and biotinylated anti-
moCD4 (20 µg/ml) or biotinylated anti-moCD28 (20 µg/ml) Ab, followed by cross-linking
with streptavidin for the time indicated in the figure.

2.3.5 Mouse models
129/Ola mice were purchased from Harlan (Indianapolis, IN). Fyn KO mice (B6;129S7-
Fyntm1Sor/J)(Stein, Lee et al. 1992) were purchased from JAX® mice and services (Bar
Harbor, MA, Stock#002385) while the conditional KO mice carrying deletion of Csk
exclusively in CD4
+
T cells has already been described(Schmedt, Saijo et al. 1998).
Thymi were isolated from 4/6-week-old mice, and thymocytes were purified using
standard protocols. All procedures involving animals described in this manuscript were
approved by the USC (protocol # 10714 and # 10853, PI: Nunzio Bottini) and LIAI
(protocol # AP-NB1-0709, PI: Nunzio Bottini) IACUC.

2.3.6 Isolation of primary human T cells and genotyping
Anonymous buffy coats were purchased from Advanced Bioservices, LLC, (Reseda, CA)
or obtained from the blood bank of the Tor Vergata University Hospital in Rome, Italy. T
cells were isolated by Lymphoprep (VWR) or Ficoll-Paque (GE Healthcare) gradient
centrifugation followed by depletion of B cells and monocytes by anti-CD19 and anti-
CD14 Dynabeads (Invitrogen). If necessary in order to induce expression of LYP, cells
were cultured in the presence of 10 ng/ml PMA for 24 hours. When needed, genomic
DNA was extracted from 100 µl of peripheral blood using a genomic DNA extraction kit
(Qiagen, Inc.) and the genotype at the LYP-R620W locus (SNP rs2476601) was
26
determined by RFLP-PCR as described in (Bottini, Musumeci et al. 2004). All
procedures involving human subjects described in this manuscript were approved by the
USC IRB (exempt approval # 053029, PI: Nunzio Bottini) or by the Ethical Committee of
the Tor Vergata University Hospital in Rome, Italy.

2.3.7 Immunoprecipitations
For IPs, cells were lysed in 20 mM Tris/HCl pH 7.4, 150 mM NaCl, 5 mM EDTA (TNE),
with 1% NP-40, 1 mM PMSF, 10 µg/ml aprotinin/leupeptin and 10 µg/ml soybean trypsin
inhibitor. The lysis buffer also contained either 5 mM iodoacetamide or Na
3
VO
4
in
concentrations variable between 1 and 10 mM. 10 mM Na
3
VO
4
was added when it was
necessary to preserve the phosphorylation of active LYP/PEP in TCR-stimulated cells.
The IP of LYP by PT66 or 4G10 Ab was not affected by the presence of Na
3
VO
4
in the
lysis buffer up to 5 or 10 mM respectively (data not shown).

2.3.8 Phospho-mass spectometry
In-gel digestion and phosphopeptide enrichment. Coomassie gel bands of interest
were excised and chopped into small pieces and digested by trypsin as described
elsewhere (Shevchenko, Wilm et al. 1996). In brief, proteins were reduced in 10 mM
DTT for 30 min at room temperature (RT), alkylated in 55 mM iodoacetamide for 30 min
at RT in the dark and digested overnight at RT with 12.5 ng/µl trypsin (Proteomics
Grade, Sigma). The digestion media was then acidified to 2% TFA. The supernatant was
loaded onto a self-packed 200 µl pipette tip plugged with C18 material (3M EmporeTM
C18 disk, 3M Bioanalytical Technologies, St. Paul, MN) filled first with 2 mm of TiO
2

beads (GL Sciences Inc. Tokyo, Japan) and then another C18 disk. Bound peptides to
the first upper C18 disk were desalted by a wash with 0.1% TFA and then eluted onto
27
underneath TiO
2
beads with 30mg/ml DHB in 80% ACN and 0.1% TFA. The bound
peptides to TiO
2
were washed 1x with the previous buffer and then 1x with a similar
buffer without DHB. Peptides were eluted using 50ul of 20% NH
4
OH in 40% ACN in
water, pH ≥10.5. Phosphopeptide mixture was almost dried using a SpeedVac
concentrator (Concentrator 5301, Eppendorf AG, Hamburg Germany) and then re-
suspended in 0.1% of TFA for LC-MS/MS analysis.
Mass spectrometry analysis. An LTQ-Orbitrap mass spectrometer (ThermoElectron,
Bremen, Germany) coupled online to nano-LC (Ultimate, Dionex, USA) was used. To
prepare an analytical column C
18
material (ReproSil-Pur C18-AQ 3 µm; Dr. Maisch
GmbH, Ammerbuch-Entringen, Germany) was packed into a spray emitter (75 µm ID, 8
µm opening, 70 mm length; New Objectives, USA) using a high-pressure packing device
(Nanobaume
TM
, Western Fluids Engineering, USA). Mobile phase A consisted of water,
5% acetonitrile and 0.5% acetic acid and mobile phase B of acetonitrile and 0.5% acetic
acid. The five most intense peaks of the MS scan were selected in the ion trap for MS
2
,
(Normal scan, filling 5x10
5
ions, maximum fill time 500ms for MS scan, 2x10
5
ions for
MS
2
, multistage activation enabled, maximum fill time 200 ms, dynamic exclusion for 60
seconds). Raw files were processed using DTAsupercharge v.1.18
(http://msquant.sourceforge.net/). The generated peak lists were searched against the
IPI human database using Mascot 2.2 with the parameters: monoisotopic masses, 10
ppm on MS and 0.5 Da on MS/MS, ESI TRAP parameters, full tryptic specificity,
cysteine carbamidomethylated as fixed modification, oxidation on methionine,
phosphorylation on serine, threonine, tyrosine, protein N-acetylation and deamidation on
glutamine and asparagine as variable modifications, three missed cleavage sites
allowed. The results were parsed through MSQuant 1.4.3a74
(http://msquant.sourceforge.net/). Identified phosphopeptide was manually validated.
28
2.3.9 Luciferase assays
Luciferase assays were performed as described in(Vang, Congia et al. 2005). The
difference in the ratio between firefly and Renilla luciferase activity in stimulated versus
unstimulated cells (TCR-induced increased activation of reporter) was then plotted
against the expression of LYP assessed by densitometric scanning of anti-HA blots of
total lysates.

2.3.10 Flow cytometry assays
All samples were acquired on a FACSCanto II (BD Biosciences). Data were analyzed
using FlowJo software (TreeStar, Ashland, OR). For induction of CD69, cells were
stimulated with 5 µg/ml OKT3 for 4 hours at 37 °. Cells were then washed, fixed,
permeabilized, blocked with 10% mouse serum and costained with AlexaFluor488
(AF488)-conjugated anti-HA antibody (Cell Signaling Technology) and APC-conjugated
anti-CD69 antibody. Cells overexpressing LYP were gated by comparing AF488
fluorescence of cells transfected with HA-LYP versus cells transfected with vector alone.

2.3.11 Phosphatase assays using a novel peptidic fluorogenic substrate
Autodephosphorylation of PTPs is a well-known phenomenon, which complicates the
assessment of the effect of phosphorylation on the phosphatase activity of PTPs. We
observed initial autodephosphorylation of immunoprecipitated LYP already after 5
minutes of incubation in phosphatase buffer (Bis-Tris pH 6.0, 5 mM DTT) (data not
shown). To study the activity of phospho-LYP in conditions of fast enzyme
autodephosphorylation, we used a fluorogenic peptide based on a pTyr mimicking
coumarin amino acid, which has recently been developed by the Barrios group (Mitra
and Barrios 2005). The enantiomerically pure, appropriately protected, phosphorylated
29
coumaryl amino propionic acid (pCAP) can be incorporated into peptide substrates using
standard FMOC-based solid phase peptide synthesis (SPPS) methodologies and
undergoes enzymatic dephosphorylation by PTPs to CAP (Mitra and Barrios 2005).
Upon excitation around 340 nm, CAP-containing peptides are over 10
4
times more
fluorescent than pCAP-containing peptides ( λ
em
=460 nm). Thus, PTP-catalyzed
hydrolysis of pCAP-containing peptides results in a fluorogenic, continuous and direct
assay for PTP activity. The assay is extremely sensitive compared to the standard pTyr
peptide assay, and minimal spontaneous hydrolysis of the fluorogenic peptide ensures
that the signal/background ratio of this assay is optimal even for short assay times (Mitra
and Barrios 2005; Mitra and Barrios 2007). Also because the assay is direct, continuous
monitoring of linearity of the reaction is possible. We synthesized and purified the pCAP-
containing peptide substrate ARLIEDNE(pCAP)TAREG (peptide 14LckpCAP394), a
sequence based on residues around the Lck Tyr394 autophosphorylation site, which is a
physiological substrate of LYP. A shorter version of this peptide has been reported to be
an excellent LYP substrate (Mitra and Barrios 2007) and we found that the recombinant
catalytic domain of LYP dephosphorylates the 14LckpCAP394 peptide following
Michaelis-Menten kinetics, and with K
M
and k
cat
equal or better than the corresponding
14 aa pTyr peptide (k
cat
was 11.7 sec
-1
and 4.4 sec
-1
and K
M
was 81 µM and 71 µM for
the pCAP and pTyr peptides respectively, our unpublished data). For detection of
phosphatase activity of LYP IPed from transfected cells, the IPs were washed in Bis-Tris
pH 6.0, and then resuspended in phosphatase buffer (Bis-Tris pH 6.0, 5 mM DTT). After
addition of 0.4 mM peptide, the reaction was monitored continuously by measuring the
increase in fluorescence ( λ
ex
= 340 nm and λ
em
= 460 nm) at 60 s intervals for 30 min.
The activity measured in triplicate was corrected for the nonspecific signal of identical
reactions performed also in triplicate without addition of enzyme. The activity corrected
30
for background fluorescence of substrate alone was then normalized for LYP expression
as assessed by anti-HA WB of fractions of IPs taken before resuspension in the final
phosphatase buffer.

2.3.12 Graphs and Statistics
Graphs, curve fittings and kinetic parameter calculations were performed using the
Graphpad Prism software package (Graphpad, San Diego, CA). All SD of differences
and ratios were calculated according to the error propagation rules described by Taylor
(Taylor 1997).

2.4 Results
2.4.1 Binding of Csk to LYP does not directly affect the phosphatase activity
To explain the gain-of-function phenotype of LYP-W620, we first assessed whether i) the
polymorphism directly affects the protein to induce increased enzymatic activity, or ii)
simple binding of LYP to Csk is sufficient to inhibit the activity of the phosphatase. We
purified recombinant full-length LYP-R620 and LYP-W620 and their inactive C227S
mutants from lysates of insect cells (Fig. 2.1A). Because insect Csk lacks a functional
SH3 domain (Read, Bach et al. 2004), we obtained Csk-free LYP, which could be used
to reconstitute in vitro the complex between LYP and Csk (Fig. 2.1B). When we
measured the activity of recombinant LYP using an Lck-derived peptide as a substrate,
we observed no significant difference between the activity of LYP-R620 and LYP-W620
(Fig. 2.1C). In control assays both LYP-R620 and LYP-W620 -C227S variants did not
show any activity (data not shown). The addition of Csk to the reaction did not affect the
activity of LYP-R620 or LYP-W620 (Fig. 2.1C). Despite the limitations imposed by our in
vitro system (for example, differences in post-translational modifications of LYP between
31
mammalian and insect cells), these data suggest that the polymorphism does not induce
gain-of-function through a direct effect on the protein, and that binding of Csk to LYP is
not sufficient per se to induce changes in the enzymatic activity.

32
Figure 2.1. Binding of Csk to LYP does not directly affect the phosphatase activity. (A,B) In vitro
reconstitution of the LYP/Csk complex. (A) Isolation of recombinant full-length LYP-R620 and LYP-W620.
The figure shows a silver stained polyacrylamide gel with 300 ng of purified LYP-R620 (lane 1), LYP-
R620/S227 (lane 2), LYP-W620 (lane 3), or LYP-W620/S227 (lane 4). The arrow indicates LYP, and the
arrowhead indicates a non-specific protein which co-purifies with LYP in our preparation. (B) LYP-R620
binds Csk more efficiently than LYP-W620. 25 ng recombinant LYP-R620 (lanes 1-5) or LYP-W620 (lanes 6-
10) bound to M2-FLAG beads were incubated with increasing amount of recombinant His-Csk in 20 mM
Tris/HCl, pH 7.4, 150 mM NaCl and 1 mM EDTA overnight at 4°C. The complex was washed twice with the
same buffer and run on a polyacrylamide gel. (C) Binding to Csk does not directly affect the activity of LYP.
The activity of 2 ng recombinant LYP-R620 (squares) or LYP-W620 (triangles) was assayed in the presence
(open symbols, dotted lines columns) or absence (filled symbols, continuous lines) of 60 ng recombinant
His-Csk using 0.4 mM 14LckpCAP394 peptide as substrate in 50 mM Bis-Tris pH 6.0, 1mM DTT. Diamond
symbols and dashed line show fluorescence of control reaction carried out without adding any enzyme. The
reaction was followed continuously in order to ensure initial rate conditions. Symbols show average ± SD
activity at various incubation time. Regression lines are shown. Significance of the differences has been
calculated using ANOVA. Identical results were obtained when the assays on LYP-R620 were repeated
using a 50 mM Tris-HCl, 1 mM DTT, pH 7.4 buffer (data not shown). Experiments performed by Yingge Liu.

33
2.4.2 TCR stimulation induces tyrosine phosphorylation of LYP
We next hypothesized that differences in post-translational modifications between LYP-
R620 and LYP-W620 contribute to the gain-of-function phenotype of LYP-W620. Given
that LYP forms a complex with Csk, a PTK, and the polymorphism affects the PTP/PTK
interaction, we considered the possibility that the phosphatase could be phosphorylated
on tyrosine. Several PTPs are regulated by tyrosine phosphorylation(Tailor, Gilman et al.
1997; Lu, Gong et al. 2001), including PTP20(Aoki, Ueno et al. 2004), which is homolog
to LYP. We found that treatment of human Jurkat-large T Antigen T cells [JTAg] (Shaw,
Utz et al. 1988) with pervanadate, a powerful PTP inhibitor, induced phosphorylation of
LYP on tyrosine (Fig. 2.2A). TCR engagement also caused phosphorylation of LYP in
the Jurkat E6.1, JTAg and Hut78 T cell lines (Fig. 2.2B, Fig. 2.2C and data not shown)
and in primary human T cells (Fig. 2.2D). The kinetics of LYP phosphorylation were fast
(in JTAg cells it was detected after 15” stimulation, data not shown), and peaked
between 1 and 2 minutes (Fig. 2.2B, Fig. 2.2C, and Fig. 2.2D). Endogenous PEP was
also tyrosine-phosphorylated after TCR stimulation of mouse thymocytes (Fig. 2.2E).
Transfected HA-tagged LYP and PEP were similarly phosphorylated after TCR-
engagement in JTAg (Fig. 2.2F and data not shown). Inactive PTP mutants are often
used to study their regulation by phosphorylation on tyrosine residues, because PTPs
often show a strong tendency towards auto-dephosphorylation (Tailor, Gilman et al.
1997; Aoki, Ueno et al. 2004). Indeed, we observed that TCR-induced phosphorylation
of transfected inactive C227S mutants of LYP and PEP was several-fold more prominent
than that of the wild-type phosphatases (data not shown). The identity of anti-phospho-
tyrosine [pTyr]-reactive LYP and PEP immunoprecipitated [IPed] from lysates of
transfected T cells was confirmed by using an S-tagged mutant of LYP and PEP (Fig.
2.2F and data not shown).
34
Figure 2.2. TCR-induced tyrosine phosphorylation of LYP. (A) PV treatment of JTAg cells induces LYP
phosphorylation. LYP was IPed from lysates of JTAg cells left unstimulated (lanes 1 and 2) or treated with
200 μM PV for 15’ (lanes 3 and 4). Cell lysates were subjected to IP using an anti-LYP Ab (lanes 1 and 3) or
to control precipitation using beads alone (lanes 2 and 4). (B-D) TCR stimulation induces LYP
phosphorylation. (B) LYP was IPed from lysates of JTAg cells left unstimulated (lane 1) or stimulated with
C305 for 1’, 2’, or 5’ (lanes 2-4). (C) Anti-pTyr IPs were performed from lysates of JTAg cells left
unstimulated (lane 1) or stimulated with C305 to stimulate the TCR for 2’ (lane 2). Performed by Valeria
Orrú. (D) IP of LYP from lysates of primary human T cells left unstimulated (lane 1) or stimulated with anti-
CD3+anti-CD4 for 40” (lane 2) or 90” (lane 3). Performed by Stephanie Stanford and Edoardo Fiorillo. (E)
TCR-induced Tyr phosphorylation of PEP. Anti-pTyr IPs were performed from lysates of primary mouse
thymocytes left unstimulated (lane 1) or stimulated with anti-CD3+anti-CD4 for 2’ (lane 2). Similar results
were obtained in additional sets of experiments which were performed by IPing PEP and blotting IPs with
anti-pTyr Ab and stimulating cells with anti-CD3+anti-CD4 or anti-CD3+anti-CD28 (data not shown).
Performed by Valeria Orrú. (F) TCR stimulation of JTAg cells induces phosphorylation of transfected LYP.
JTAg cells were transfected with the inactive C227S mutant of LYP-R620 (lanes 1-4) or with a construct
expressing the same mutant in fusion with an additional N-terminal 15 aa S-tag (lanes 5-8, the S-tag slows
the migration of HA-LYP on polyacrylamide gels). Cells were left unstimulated (lane 1,5) or stimulated with
C305 for 1’ (lanes 2,6), 2’ (lanes 3,7), or 5’ (lanes 4,8). Black arrows indicate the position of HA-LYP or HA-
S-LYP. Performed by Edoardo Fiorillo. Similar results were obtained in additional experiments where JTAg
cells were transfected with an inactive C227S mutant of PEP in fusion or not with an amino-terminal 15 aa
S-tag (data not shown).

35
2.4.3 Autoimmune-associated LYP-R620W polymorphism affects TCR-induced
phosphorylation of LYP
Next, we assessed whether LYP-R620 and LYP-W620 show any difference in TCR-
induced phosphorylation. TCR stimulation induced much higher phosphorylation of LYP-
R620 than of LYP-W620 in JTAg and Jurkat E6.1 cells (Fig. 2.3A and data not shown).
Phosphorylation of LYP-R620 in resting cells was also higher than LYP-W620 (data not
shown). The difference in phosphorylation between R620 and W620 was independent of
the expression levels of the two LYP variants, and was observed even at very low over-
expression levels (data not shown). Similar results were obtained when the two homolog
variants of PEP (PEP-R619 and PEP-W619) were transfected in JTAg cells (data not
shown). Fig. 2.3B shows that LYP IPed from primary T cells isolated from healthy
subjects of RR genotype have greater phosphorylation than LYP IPed from cells of RW
genotype. We concluded that LYP and PEP are phosphorylated on tyrosine at levels that
are detectable in resting T cells and are strongly induced in the early phase of TCR
signaling. In transfected cells LYP-R620 also has greater phosphorylation on tyrosine
than LYP-W620, in resting and stimulated T cells. We have preliminary evidence that
this is true also in primary cells, although more experiments are warranted, especially
comparing subjects of RR genotype with subjects of WW genotype.

36
Figure 2.3. LYP-W620 is less phosphorylated in resting and TCR-stimulated T cells. (A) IP from transfected
JTAg cells. Anti-HA IPs were performed from lysates of JTAg cells transfected with LYP-R620 (lanes 1-4) or
LYP-W620 (lanes 5-8). Cells were left unstimulated (lanes 1,5) or stimulated with C305 for 1’ (lanes 2,6), 2’
(lanes 3,7), or 5’ (lanes 4,8). The efficiency of TCR stimulation was similar in LYP-R620 and LYP-W620
transfected cells, as shown by anti-pZAP70(Y319) and anti-ZAP70 blots of total lysates. Performed by
Edoardo Fiorillo. (B) LYP-W620 is less phosphorylated than LYP-R620 in resting T cells. IP of endogenous
LYP from primary human T cells from healthy subjects of RR (lane 1) or RW (lane 2) genotype. The
observation was replicated on an additional couple of unrelated control subjects of RR and RW genotype.
Performed by Edoardo Fiorillo and Novella Rapini.

37
2.4.4 Lck phosphorylates LYP in T cells
Next, we set out to assess which PTK is responsible for the phosphorylation of LYP in T
cells. Incubation of JTAg cells with 10 µM of the Src-family kinase inhibitor PP2
completely abolished the basal and TCR-induced phosphorylation of endogenous LYP
(data not shown), suggesting that phosphorylation of LYP is dependent on the activity of
Src-family PTKs. When we co-transfected LYP with a set of candidate PTKs in COS
cells, we observed that Lck, Fyn, and Csk could phosphorylate LYP, while ZAP70 and
Itk could not (Fig. 2.4A). Lck was the most efficient LYP kinase in this assay and in
additional in vitro kinase assays (Fig. 2.4B). RNAi-mediated knock-down of Lck and Csk
in T cells respectively abolished and reduced TCR-induced tyrosine phosphorylation of
endogenous LYP (Fig. 2.4C and data not shown), while knock-down of Fyn did not
seem to substantially affect LYP phosphorylation (Fig. 2.4D). The phosphorylation of
PEP was conserved in Fyn
-/-
(Stein, Lee et al. 1992) thymocytes (Fig. 2.4E), further
arguing against a role of Fyn as the major LYP kinase in T cells. On the other hand, Fig.
2.4F shows that the phosphorylation of PEP was reduced but not abolished in
thymocytes isolated from mice carrying a conditional deletion of Csk in CD4
+
cells. In
these experiments all the TCR-expressing thymocytes were virtually Csk KO. Assuming
the absence of significant compensatory mechanisms in the cells analyzed, these data
suggest that Lck and Csk are the major mediators of TCR-induced tyrosine
phosphorylation of LYP in T cells, although we cannot formally exclude the role of other
kinases.

38
Figure 2.4. Phosphorylation of LYP in T cells depends on Csk and Lck. (A) Kinase screening in COS cells.
COS cells were co-transfected with LYP-R620/S227 and Lck (lane 1), Fyn (lane 2), Csk (lane 3), ZAP70
(lane 4), Itk (lane 5), or empty vector (lane 6) and total lysates were subjected to denaturing anti-pTyr IP.
Similar results were obtained in separate experiments, performed by IPing LYP and blotting with anti-pTyr
Ab (data not shown). Performed by Valeria Orrú. (B) In vitro phosphorylation of LYP by PTKs. LYP-
R620/S227 was IPed from transfected COS cells and in vitro phosphorylated with recombinant Csk (lane 2),
Fyn (lane 3) or Lck (lane 4). Lane 1 is a control reaction without PTK. Performed by Edoardo Fiorillo. (C,D)
Phosphorylation of LYP is reduced by knock-down of Lck and Csk, but is not affected by knock-down of Fyn.
(C) Endogenous LYP was IPed from lysates of JTAg cells transfected with RNAi oligos specific for Lck
(lanes 1,2), Csk (lanes 5,6) or media alone (lanes 3,4). Cells were left unstimulated (lanes 1, 3, 5) or
subjected to 2’ stimulation with C305 (lanes 2,4,6). Similar results were obtained by performing denaturing
anti-pTyr IPs followed by WB with anti-LYP Ab (data not shown). Performed by Edorado Fiorillo. (D)
Endogenous LYP was IPed from lysates of JTAg cells transfected with a non-targeting oligo (lanes 1 and 2)
or an oligo specific for Fyn (lanes 3 and 4). Cells were left unstimulated (lanes 1 and 3) or subjected to 2’
stimulation with C305 (lanes 2 and 4). (E) PEP phosphorylation is conserved in Fyn
-/-
thymocytes. Anti-PEP
IPs were performed from lysates of thymocytes isolated from Fyn
-/-
mice (lanes 3 and 4) and from wild-type
littermates (lanes 1 and 2). Cells were left unstimulated (lanes 1 and 3) or stimulated with anti-CD3+anti-
CD4 for 1’ (lanes 2 and 4). Identical results were obtained by performing anti-PEP blot of anti-pTyr IPs (data
not shown). Performed by Valeria Orrú. (F) PEP phosphorylation is reduced in T cells from Csk
-/-
mice.
Thymocytes were isolated from Csk conditional KO mice (lanes 1, 2, 5 and 6) and control littermates (lanes
3, 4, 7 and 8). Cells were left unstimulated (lanes 1-4) or stimulated with anti-CD3+anti-CD4 for 1’ (lanes 5-
6). Cell lysates were subjected to IP using an anti-Pep Ab (lanes 1, 3, 5 and 7) or to control precipitation
using normal rabbit serum (lanes 2, 4, 6, and 8).

39
2.4.5 Lck interacts with LYP in a Csk-dependent manner
We next tested whether Lck co-precipitates with a deletion mutant of LYP which is
missing the catalytic domain ( ∆288LYP, including aa 289-807). This mutant was used in
order to exclude interactions due to the substrate-trapping activity of LYP (Wu, Katrekar
et al. 2006). We found that ∆288LYP is able to form a complex with Csk as well as Lck,
but not with other PTKs in T cells. Both complexes were constitutive and apparently
unaffected by TCR stimulation (Fig. 2.5A). The anti-Lck antibody could not precipitate
LYP from lysates of the Lck-negative JCaM1 cells (Straus and Weiss 1992), further
supporting the specificity of the interaction. Interestingly, the R620W mutation of
∆288LYP reduced the co-precipitation of ∆288LYP with Lck and Csk (Fig. 2.5B). The co-
precipitation between full-length LYP and Lck was decreased by the R620W mutation as
well (Fig. 2.5C). Since the effect of the R620W mutation cannot be due to a direct
interaction between the SH3 domain of Lck and the P1 domain of LYP ((Cloutier and
Veillette 1996) and our unpublished observation), we assessed whether Csk affects the
interaction between LYP and Lck. Fig. 2.5D shows that knock-down of Csk in T cells
reduced the co-precipitation of LYP with Lck, and abolished the difference between LYP-
R620 and LYP-W620. Co-precipitation between Lck and PEP was also much decreased
in thymocytes from Csk conditional KO mice (Fig. 2.5E). It is unlikely the results shown
in Fig. 2.5D and 2.5E are due to decreased trapping of Lck by LYP, since knock-
down/knockout of Csk rather leads to increased phosphorylation of Lck on
Y394(Schmedt, Saijo et al. 1998; Vang, Abrahamsen et al. 2004). The above-mentioned
data supports a model where recruitment of Csk to the P1 domain of LYP facilitates i)
the interaction between LYP and Lck and ii) the phosphorylation of LYP on tyrosine
residue(s). Reduced binding of Csk to LYP-W620 leads to reduced recruitment of Lck to
the LYP protein complex and reduced phosphorylation of LYP-W620. In support of our
40
model we also observed that i) there was no significant in vitro co-precipitation between
recombinant LYP and Lck purified from insect cell lysates (data not shown); ii) a C-
terminal truncation of LYP at aa 517 effectively abolished any interaction between LYP
and Lck in T cells and in co-transfected COS cells (data not shown); and iii) LYP-R620
and LYP-W620 purified from insect cell lysates showed identical low level of
phosphorylation on tyrosine, as assessed by Western blotting [WB] using an anti-pTyr
Ab (data not shown). The mechanism of Csk-mediated recruitment of Lck to LYP is
unclear, however, as shown in Fig. 2.5F, overexpression of a kinase-dead mutant of Csk
reduced co-precipitation between Lck and LYP, suggesting that recruitment of Lck
depends at least in part on the kinase activity of Csk.

41
Figure 2.5. Csk-dependent co-precipitation of Lck with LYP. (A) Lck co-precipitates with ∆288LYP. JTAg
cells were transfected with ∆288LYP and IPs were performed from lysates of resting cells (lanes 1,3,5,7,9)
or cells stimulated for 2’ with C305 (lanes 2,4,6,8,10) using an Ab against Lck (lanes 1,2), Fyn (lanes 3,4),
Itk (lanes 5,6), Csk (lanes 7,8) or ZAP70 (lanes 9,10). Peformed by Edoardo Fiorillo. (B-C) More Lck co-
precipitates with LYP-R620 than with LYP-W620. (B) JTAg cells were transfected with ∆288LYP-R620
(lanes 1,4), ∆288LYP-W620 (lanes 2,5) or empty vector (lanes 3,6) and IPs were performed from lysates of
resting cells using an Ab against Lck (lanes 1-3), or Csk (lanes 4-6). Performed by Edoardo Fiorillo. (C)
JTAg cells were transfected with LYP-R620 (lane 1), LYP-W620 (lane 2) or empty vector (lane 3) and IPs
were performed from lysates of resting cells using an anti-Lck Ab. (D-F) The co-precipitation of Lck with LYP
is dependent upon Csk activity. Performed by Edoardo Fiorillo. (D) JTAg were co-transfected with LYP-R620
(lanes 1-3 and 7-9) or LYP-W620 (lanes 4-6 and 10-12) and with RNAi oligos specific for Csk (lanes
2,5,8,11) or non-targeting ones (lanes 1,3,4,6,7,9,10,12). Cells were left unstimulated (lanes 1-6) or
stimulated 2’ with C305 (lanes 7-12). Cell lysates were subjected to IP using an anti-Lck Ab. Performed by
Edoardo Fiorillo. (E) Thymocytes were isolated from Csk conditional KO mice (lanes 2 and 4) and control
littermates (lanes 1 and 3). Cells were left unstimulated (lanes 1 and 2) or stimulated with anti-CD3+anti-
CD4 for 1’ (lanes 3 and 4). Cell lysates were subjected to IP using an anti-PEP Ab. (F) JTAg cells stably
overexpressing ∆288LYP-R620 were transfected with HA-Csk (lane 1), or decreasing amounts (3, 2, or 1 µg
of plasmid DNA) of the catalytically inactive mutant HA-Csk K222R (lanes 2-4), or vector alone (lane 5).
Cells were stimulated for 2’ with C305 and IPs were performed from lysates using an anti-Lck Ab.

42
Figure 2.5: Continued

43
2.4.6 Tyr536 is a major Lck phosphorylation site of LYP
Hypothesizing that Lck is a major LYP kinase, we set out to map the Lck
phosphorylation site(s) which are less phosphorylated in LYP-W620 than LYP-R620. We
noticed that i) N-terminal truncation of LYP up to aa 517 did not significantly affect the
phosphorylation of LYP by Lck in COS cells (Fig. 2.6A), and ii) the difference in TCR-
induced phosphorylation between the two LYP variants was conserved after N-terminal
truncation of the protein to aa 517 (Fig. 2.6B). We also noticed that transfected LYP2
was efficiently phosphorylated in T cells (data not shown). Because LYP2 lacks the last
three C-terminal polyproline domains between aa 685-807 (Cohen, Dadi et al. 1999), we
reasoned that a major Lck phosphorylation site affected by the R620W variation is likely
located between aa 518 and 684. Thus we mutagenized all five Tyr residues between aa
518 and 684 into Phe. Fig. 2.6C and 2.6D show that Y536F was the only mutation which
abolished phosphorylation of ∆517-LYP-R620 by Lck in COS cells and the TCR-induced
phosphorylation of ∆517 -LYP-R620 in T cells. Interestingly, in the region between aa
518-684, Y536 is one of the only two Tyr residues which are highly conserved among
human, mouse, rat, and cow, and the only one surrounded by a highly conserved aa
motif (Fig. 2.6E). Analysis by Netphos (http://www.cbs.dtu.dk/services/NetPhos/) (Blom,
Gammeltoft et al. 1999) also indicated Tyr536 as the only putative Lck phosphorylation
site in the aa 518-684 region (data not shown). Phospho-mass spec analysis of
recombinant LYP-S227 in vitro phosphorylated with Lck detected phosphate on Tyr536
(Fig. 2.7A), further supporting the idea that the 536 residue is a direct phosphorylation
site for Lck. Phosphorylation of the Y536F mutant of full-length LYP by Lck in COS cells
was dramatically decreased (Fig. 2.7B). We concluded that Tyr536 is a major Lck
phosphorylation site in LYP. As mentioned, reduced phosphorylation of LYP-W620 by
Csk could also play a role in the mechanism of action of the R620W mutation. However,
44
N-terminal truncation of LYP at aa 517 abolished the phosphorylation of LYP by Csk in
COS cells, suggesting that Tyr536 is not a Csk phosphorylation site (Fig. 2.7C).
45
Figure 2.6. Tyr536 is a major Lck phosphorylation site of LYP. (A,B) Mapping by use of truncation mutants.
(A) Anti-FLAG IPs were performed from lysates of COS cells transfected with FLAG-tagged full-length LYP-
S227 (lanes 1 and 5) or the truncation mutants ∆288LYP (lanes 2 and 6), ∆399LYP (lanes 3 and 7),
∆517LYP (lanes 4 and 8) alone (lanes 5-8) or together with Lck (lanes 1-4). Peformed by Valeria Orrú. (B)
JTAg cells were transfected with FLAG-tagged truncation mutants ∆517LYP-R620 (lanes 1, 3, 5) or
∆517LYP-W620 (lanes 2,4,6). Lanes 1-4: denaturing IPs were performed from lysates of unstimulated cells
(lanes 3 and 4) or cells stimulated with C305 for 2’ (1 and 2), using anti-pTyr Ab. Lanes 5-6 show total
lysates. (C,D) Mapping of Tyr536 by site-specific mutagenesis. Performed by Lucia Delogu. (C) Anti-FLAG
IPs were performed from lysates of COS cells transfected with FLAG-tagged truncation mutants ∆517LYP-
F526 (lanes 1 and 2), ∆517LYP-F528 (lanes 3 and 4), ∆517LYP-F536 (lanes 5 and 6), ∆517LYP-F577
(lanes 7 and 8), ∆517LYP-F578 (lanes 9 and 10), or ∆517LYP-WT (lanes 11 and 12) alone (lanes
2,4,5,7,9,11) or together with Lck (lanes 1,3,6,8,10,12). Performed by Valeria Orrú. (D) JTAg cells were
transfected with FLAG-tagged truncation mutants ∆517LYP-F526 (lanes 1 and 2), ∆517LYP-F528 (lanes 3
and 4), ∆517LYP-F536 (lanes 5 and 6), ∆517LYP-F577 (lanes 7 and 8), ∆517LYP-F578 (lanes 9 and 10), or
∆517LYP-WT (lanes 11 and 12). IPs were performed from lysates of unstimulated cells (lanes 1,3,5,7,9,11)
or cells stimulated with C305 for 2’ (lanes 2,4,6,8,10,12), using anti-FLAG M2 beads. Performed by Edoardo
Fiorillo. (E) Tyr536 is located within a highly conserved motif in the interdomain of LYP. Alignment of the aa
518-684 region of human LYP (Homo sapiens) with the homolog region of LYP from cow (Bos taurus),
mouse (Mus musculus) and rat (Rattus Norvegicus). The alignment was performed using CLUSTALW2
(http://www.ebi.ac.uk/Tools/clustalw2/index.html (Chenna, Sugawara et al. 2003). Figure shows alignment in
ALN/ClustalW2 format: asterisks indicate identities, colons indicate conservative substitutions, and periods
indicate semi-conservative substitutions. Underlined Ys indicate fully conserved Tyr residues in the region.
The fully conserved motif around Tyr536 is highlighted in gray.

46
Figure 2.7. Tyr536 is a direct phosphorylation site for Lck but not for Csk. (A) Detection of pTyr536 by
phospho-mass spectrometry. Recombinant LYP-S227 was in vitro phosphorylated with Lck and the resulting
protein mixture was separated by SDS-PAGE and stained by Coomassie. The LYP protein band was
excised and digested in gel by trypsin. The resulting peptide mixture was analyzed by nanoLC-MS/MS.
Panel shows the MS/MS spectrum of the peptide spanning residues 517–548 of LYP, showing fragment
ions that unambiguously pinpoints phosphorylation of residue Tyr536. Peformed by Edoardo Fiorillo and
Mogjiborahman Salek (B) Tyr536 is a major Lck phosphorylation site. Anti-HA IPs were performed from
lysates of COS cells transfected with LYP-WT and LYP-F536 alone (lanes 1 and 2) or with Lck (lanes 3 and
4). Perfomed by Valeria Orrú. (C). Tyr536 is unlikely to be a Csk phosphorylation site. Anti-FLAG IPs were
performed from lysates of COS cells transfected with Csk together with FLAG-tagged full-length LYP-S227
(lane 1) or the truncation mutants ∆288LYP (lane 2), ∆399LYP (lane 3), ∆517LYP (lane 4). Performed by
Valeria Orrú.

47
2.4.7 Tyr536Phe mutation of LYP-R620 induces gain-of-function activity
Fig. 2.8A shows that in T cells mutation of Tyr536 to Phe reduced the difference in
phosphorylation between LYP-R620 and -W620. However, the Tyr536Phe mutation did
not completely eliminate either the overall phosphorylation of LYP or the difference in
phosphorylation between the two variants of LYP, suggesting that there is at least one
additional site which is less phosphorylated in LYP-W620. Next, we assessed whether
the mutagenesis of Tyr536 into a Phe in LYP-R620 could mimic at least in part the gain-
of function phenotype of LYP-W620. LYP-R620/F536 inhibited TCR signaling more
efficiently than the wild-type LYP-R620, as assessed by TCR-induced activation of an
NFAT/AP1 luciferase reporter and induction of CD69 expression (Fig. 2.8B and 2.8C).
These data also show that the F536 mutation attenuated differences in TCR signaling
inhibition between LYP-R620 and LYP-W620 in JTAg cells. Importantly, Fig. 2.8D shows
that the Y536F mutation of LYP-R620 also led to increased LYP phosphatase activity.
We concluded that Lck-mediated phosphorylation of LYP on Tyr536 plays an inhibitory
role on the phosphatase activity, and that reduced phosphorylation on Tyr536 leads to a
gain-of-function which contributes to the phenotype shown by the autoimmune-
predisposing LYP-W620. Reduced phosphorylation of LYP-W620 at additional sites
which are targets of Lck or other kinase activities might well contribute to the same
phenotype and should be investigated.

48
Figure 2.8. Reduced phosphorylation of Tyr536 on LYP-W620 leads to gain-of-function inhibition of TCR
signaling. (A) Tyr536 is more phosphorylated in LYP-R620 than LYP-W620. Anti-HA IPs were performed
from lysates of JTAg cells transfected with LYP-R620 (lanes 1 and 2), LYP-W620 (lanes 3 and 4), LYP-
R620/F536 (lanes 5 and 6) or LYP-W620/F536 (lanes 7 and 8) constructs. Cells were left unstimulated
(lanes 1,3,5,7) or stimulated with C305 for 2’ (lanes 2,4,6,8). Performed by Edoardo Fiorillo. (B-D)
Phosphorylation on Tyr536 inhibits LYP in TCR signaling. (B) Activation of an NFAT/AP1 reporter. JTAg
cells were co-transfected with a 3xNFAT/AP1 firefly luciferase reporter, a control Renilla luciferase reporter,
and LYP-R620, LYP-W620, LYP-R620/F536, or LYP-W620/F536. Cells were stimulated for 7 h with OKT3,
then lysed and luciferase activity was measured on lysates. The average ± SD stimulation-induced increase
in the ratio between firefly and Renilla luciferase activities of lysates of cells transfected with LYP-R620 (red
squares and line), LYP-W620 (blue triangles and line), LYP-R620/F536 (green circles and line) or LYP-
W620/F536 (yellow squares and line) was plotted versus LYP expression in same lysates as assessed by
anti-HA blot. Lines are non-linear fitting of data to an exponential decay equation, and 90% confidence
intervals are shown (dashed lines). Data is representative of two experiments with similar results. Performed
by Edoardo Fiorillo. (C) Induction of CD69. JTAg cells were transfected with HA-LYP-R620, HA-LYP-W620,
HA-LYP-R620/F536, HA-LYP-W620/F536, HA-LYP-R620/S227 or vector alone. Cells were left unstimulated
or stimulated with OKT3 for 4 hours and were co-stained with an AlexaFluor488 (AF488)-conjugated anti-HA
antibody and an APC-conjugated anti-CD69 antibody. Live cells were gated by forward and side scatter and
further gated for CD69 expression by comparison with the lower half of activated cells transfected with
catalytically inactive LYP (HA-LYP-R620/S227). The corresponding percent of gated T cells is shown in
each box. Levels of overexpression of LYP mutants are shown as histograms of AF488 fluorescence of HA-
positive cells transfected with HA-LYP-R620 (red), HA-LYP-W620 (blue), HA-LYP-R620/F536 (green), HA-
LYP-W620/F536 (yellow) or HA-LYP-R620/S227 (black). Gray shaded graph shows HA-negative cells. (D)
Phosphorylation on Tyr536 inhibits the phosphatase activity of LYP. JTAg cells were transfected with HA-
LYP-R620, HA-LYP-W620 or HA-LYP-R620/F536. Anti-HA IPs were performed from lysates of cells
stimulated with C305 for 2’ in order to maximize phosphorylation of LYP, and phosphatase activity was
assessed continuously using the 14LckpCAP394 peptide as substrate. Histogram shows average ± SD
activity of IPed LYP-R620 (red column), LYP-W620 (blue column), or LYP-R620/F536 (green column)
normalized for LYP expression by densitometric scanning of anti-HA blots of fractions of the IPs (see right
panel). The time of reaction was optimized in order to ensure initial rate conditions and avoid any significant
49
auto-dephosphorylation of the phosphatase (data not shown). Data is representative of two experiments with
identical results.
Figure 2.8: Continued

50
2.5 Discussion
PTPN22 is currently classified as a “shared-autoimmunity gene”, and its association with
human autoimmunity is robust and population-independent(Bottini, Vang et al. 2006). In
Caucasian populations the contribution of PTPN22 to the genetic risk of autoimmunity is
substantial: PTPN22 currently ranks in third place (after the HLA and the Insulin genes)
and in second place (after the HLA) in terms of single-gene contribution to the etiology of
T1D and RA respectively(Todd, Walker et al. 2007).

The R620W polymorphism does not result in alterations of PTPN22 mRNA levels in
primary T cells(Nielsen, Barington et al. 2007) and the increased phosphatase activity is
so far the only known functional consequence of the R620W genetic variation. Here we
report the first study of the functional effects of the autoimmune-associated LYP-R620W
variation at the molecular level. Our current working model is summarized in Fig. 2.9,
and is based on several observations, which are discussed in detail below.

First, we found that LYP is phosphorylated on tyrosine at levels that are detectable in
resting T cells and are strongly induced in the early phase of TCR signaling. We believe
that Lck is a major LYP kinase in T cells, based on the following evidence: i) knock-down
of Lck reduced the phosphorylation of LYP; ii) Lck was an efficient LYP kinase in vitro;
iii) ZAP70 and Itk did not phosphorylate LYP, while phosphorylation of LYP was
conserved in Fyn KO cells. Other kinases might well phosphorylate LYP on tyrosine
residues in T cells, the most obvious candidate being Csk. Indeed, we also observed a
reduction of LYP phosphorylation after Csk knock-down, and in Csk KO cells.

51
Second, we showed that Lck forms a complex with LYP. This complex seems to be
constitutive, although more experiments are needed in order to exclude an effect of TCR
stimulation on the interaction between Lck and LYP. We believe that the interaction
between LYP and Lck is dependent upon/facilitated by recruitment of Csk to the P1
domain of LYP, and in turn facilitates the phosphorylation of LYP by Lck. This model is
supported by the following evidence: i) a truncation mutant of LYP missing the P1-P4
domains did not show any interaction with Lck; ii) knock-down of Csk reduced the
interaction of LYP with Lck and the phosphorylation of LYP; iii) importantly, in T cells
LYP-W620 showed reduced interaction with Lck and reduced basal and TCR-induced
phosphorylation. Further investigation at the molecular level is needed in order to assess
how Csk recruits Lck to LYP and whether Lck interacts with LYP or Csk. The data shown
in Fig. 2.5F suggests that Csk might recruit Lck to LYP by inducing phosphorylation of
LYP on a secondary site. Alternatively, it is possible that Csk recruits Lck to LYP through
additional protein interactors. Considering the known inhibitory effect of Csk on Lck
activity, Csk-mediated recruitment of Lck to LYP might contribute to keep LYP activity
high in resting cells, by increasing the dependence of LYP inhibition on TCR-induced
Lck activation levels.

Third, we mapped Tyr536 in the interdomain of LYP as one of the major Lck
phosphorylation sites and showed that phosphorylation of Tyr536 is reduced by the
R620W mutation. Our hypothesis is that reduced phosphorylation on Tyr536 contributes
to the gain-of-function phenotype of LYP-W620 and is supported by the following
evidence: i) a Tyr536Phe mutant of LYP-R620 showed gain-of-function inhibitory activity
on TCR signaling and ii) the Tyr536Phe mutation increased the phosphatase activity of
LYP-R620 to levels close to LYP-W620. Little is currently known about the structure and
52
function of the LYP interdomain, where the Tyr536 residue is located. Further
experimental evidence is needed in order to assess whether phospho-Tyr536 has a
direct effect on the activity or requires/recruits further components of the LYP protein
complex.

LYP is well known to negatively regulate Lck activity in effector T cells through
dephosphorylation of Tyr394 in the catalytic domain of the kinase (Hasegawa, Martin et
al. 2004). Our data now suggest that Lck in turn regulates LYP activity through
phosphorylation of the inhibitory Tyr536 residue in the interdomain of the phosphatase.
Mutation of Tyr536 to Phe leads to gain-of-function inhibition of T cell activation,
supporting the idea that reciprocal inhibition between LYP and Lck is an important
modulator of TCR signaling. The data suggest that after TCR engagement, increased
phosphorylation of LYP by Lck acts as a positive feedback loop, by further boosting the
activation of Lck (Fig. 2.9A). By reducing tonic inhibition of signaling by LYP at the time
of the initial wave of kinase activation, this feedback system might be critically important
in order to ensure correct TCR signal propagation. Moreover, in resting T cells the
phosphorylation levels of LYP could modulate the T cell activation threshold.

By interfering with the formation of the complex between LYP and Csk, the autoimmune-
associated LYP-R620W polymorphism causes reduced interaction between LYP and
Lck, with subsequent reduced phosphorylation of LYP on Tyr536 and gain-of-function
inhibition of TCR signaling (Fig. 2.9B). Such reduced feedback between Lck and LYP
likely plays a role in mediating the gain-of-function signaling inhibition of LYP-W620.
However, the contribution of other mechanisms cannot be excluded at present. First of
all, there are additional tyrosine residues which are less phosphorylated in LYP-W620
53
compared to LYP-R620. In addition, the polymorphism could affect the interaction of
LYP with unknown proteins and/or its recruitment to specific subcellular
fractions/compartments. Possible effects of the R620W variation on the activity of Csk
are also worthy of further investigation, as they could contribute to the gain-of-function
inhibition of signaling observed in carriers of LYP-W620.

In conclusion, we reported here the first molecular model for the gain-of-function
phenotype of the LYP-W620 variant. Our model is supported by several lines of
evidence, but also leaves some open questions. For example, our data suggest that Csk
induces indirect inhibition of LYP activity in T cells, a scenario in apparent disagreement
with the results of previous studies(Cloutier and Veillette 1999; Gjorloff-Wingren, Saxena
et al. 1999), which concluded that PEP and Csk act synergistically as TCR signaling
inhibitors. Functional differences between human LYP and mouse PEP could underlie
this discrepancy. For example, since Tyr536 is located in one of the regions with the
lowest overall homology between human and mouse LYP ((Cohen, Dadi et al. 1999),
see also Fig. 2.6E), it is possible that phosphorylation of Tyr536 has different effects on
the activity of LYP and PEP. Alternatively, it is possible that the stoichiometry of the
interaction between PTPN22 and Csk is different between human and mouse cells.
Since multiple TCR signaling regulators are known to interact with the Csk-SH3 domain
in T cells(for example see (Davidson, Cloutier et al. 1997)), competition/compensation
phenomena and the relative affinity of the various protein-protein interactions involved
should be taken into account when interpreting results obtained in over-expression
systems.

54
Figure 2.9. Model of regulation of LYP and Lck activity by reciprocal negative feedback. By Edoardo Fiorillo
and Nunzio Bottini.
55
Chapter 3: Non-phospho-mimetic inhibitors of lymphoid tyrosine phosphatase
3.1 Abstract
The lymphoid tyrosine phosphatase LYP, encoded by the gene PTPN22, is a negative
regulator of signaling through the T cell receptor. Genetic and functional evidence in
humans suggests that LYP is a drug target for autoimmunity. Here, we screened a
library of 4000 compounds with high drug-like properties, and identified a series of non-
phospho-mimetic LYP inhibitors. Upon further testing, we identified one compound which
inhibited LYP and increased early T cell signaling and T cell activation.

3.2 Introduction
Autoimmune diseases are common diseases of complex etiology, in which a
combination of genetic and environmental factors cause a loss of central and/or
peripheral immune tolerance, leading to an attack against self-antigens(Carlton, Hu et al.
2005). Signaling through the T cell receptor (TCR) plays an important role in several
mechanisms of tolerance, including thymic selection and the generation/function of
regulatory T cells(Wilkinson, Downey et al. 2005). Thus regulators of TCR signaling are
currently important candidate genes and drug targets for human autoimmunity.

Protein tyrosine phosphatases (PTPs) are very active mediators of TCR signaling and
anomalies of expression and/or genetic polymorphisms of PTPs are associated with
autoimmunity in mice and/or humans(Hermiston, Xu et al. 2003; Vogel, Strassburg et al.
2003; Siminovitch 2004; Bottini, Vang et al. 2006). PTPs are also promising drug targets
in human autoimmunity(Beers, Malloy et al. 1997; Urbanek, Suchard et al. 2001;
Hermiston, Xu et al. 2003). Among PTPs which regulate TCR signaling, the lymphoid
tyrosine phosphatase (LYP), encoded by the gene PTPN22, recently emerged as a
56
major risk factor and candidate drug target for human autoimmunity. LYP belongs to the
sub-family of PEST-enriched tyrosine phosphatases, which includes two additional
enzymes, PTP-PEST (encoded by the PTPN12 gene), and BDP1 (encoded by the
PTPN18 gene)(Andersen, Elson et al. 2001; Alonso, Sasin et al. 2004; Veillette, Rhee et
al. 2009), and is expressed exclusively in hematopoietic cells. In T cells LYP is an
important negative regulator of signal transduction through the TCR(Cloutier and
Veillette 1999; Hasegawa, Martin et al. 2004). The mechanism of regulation of TCR
signaling by LYP includes the formation of a complex between the phosphatase and the
negative regulatory kinase Csk(Cloutier and Veillette 1996; Cloutier and Veillette 1999).

A missense polymorphism, C1858T (R620W), of the PTPN22 gene is a strong risk factor
for type 1 diabetes (T1D), rheumatoid arthritis (RA), Graves’ disease, and other
autoimmune diseases(Begovich, Carlton et al. 2004; Bottini, Musumeci et al. 2004;
Bottini, Vang et al. 2006; Lee, Rho et al. 2007). Functional studies have shown that the
C1858T polymorphism results in the substitution of an Arg with a Trp in position 620
(R620W) of the protein, which is located in the LYP-Csk interaction motif and strongly
reduces the affinity of LYP for Csk(Begovich, Carlton et al. 2004; Bottini, Musumeci et al.
2004). LYP-W620 is a gain-of-function form of the enzyme, and carriers of this variant
show reduced TCR signaling(Vang, Congia et al. 2005) (Rieck, Arechiga et al. 2007).
The mechanism of the gain-of-function phenotype and its relationship with Csk binding
remains to be clarified. Reduced signaling in T cells is believed to play a role in the
pathogenic mechanism of LYP-W620, by affecting either thymic selection, or the
development/activity of regulatory T cells(Gregersen 2005; Bottini, Vang et al. 2006;
Vang, Miletic et al. 2008). Given the gain-of-function nature of the autoimmune-
predisposing LYP-W620 variation, it has been proposed that specific small molecule
57
inhibitors of LYP would be able to correct the signaling anomalies caused by LYP-W620,
thus preventing or treating autoimmunity(Vang, Congia et al. 2005; Yu, Sun et al. 2007).
Such anti-LYP therapy would effectively correct one of the biological mechanisms of
disease, at least in subjects carrying the W620 variant. In support of the idea that the
inhibition of LYP would be beneficial in autoimmunity, we recently reported that a loss-of-
function mutation of PTPN22 plays a protective role against development of systemic
lupus erythematosus(Orru, Tsai et al. 2009). Besides their possible therapeutic
relevance, specific LYP inhibitors will also be of invaluable help in understanding the
physiological role of LYP in vivo by providing complementary information to genetic
manipulations of the PTPN22 gene in mice. In this study we set out to develop small-
molecule inhibitors of LYP by chemical library screening followed by validation of the hits
in enzyme-based and TCR signaling assays.

3.3 Experimental Procedures
3.3.1 Materials, chemicals and enzymes
All chemicals were purchased from commercial sources unless otherwise noted.
Compounds were dissolved in DMSO, and stock solutions were stored at –20 °C. The
catalytic domain of CD45 was purchased from Biomol International (Plymouth Meeting,
PA). p-nitrophenyl phosphate (pNPP) was purchased from Sigma (St. Louis, MO),
DiFMUP was purchased from Invitrogen (Carlsbad, CA), and the phospho-tyrosine (pY)
peptide ARLIEDNEpYTAREG was purchased from Celtek Peptides (Nashville, TN).

3.3.2 Antibodies and reagents
Biotinylated anti-mouse-CD3 and anti-mouse-CD4 antibodies were purchased from BD
Biosciences (San Jose, CA). The anti-CD3 was purchased from eBioscience (San
58
Diego, CA). The anti-human CD4 antibody was from Lab Vision (Fremont, CA).
Streptavidin was obtained from Sigma and the F(ab’)2 crosslinker was from Jackson
Immunolabs (West Grove, PA). The polyclonal anti-pZAP(Y319), anti-ZAP70, anti-
pLck(Y505) and anti-pSrc(Y416) antibodies were obtained from Cell Signaling
Technology (Danvers, MA), while the anti-Lck and anti-ERK2 antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA). The Anti-ACTIVE MAPK polyclonal
antibody was obtained from Promega (Madison, WI). The fluorescent-conjugated anti-
pSLP76(Y128), anti-CD69, and-CD4 and anti-CD8 antibodies were purchased from BD
(Carlsbad, CA). The nitrocellulose membrane, HRP-conjugated secondary antibodies,
and ECL-Plus Chemiluminescence kit were obtained from GE-Healthcare Bio-Sciences
(Piscataway, NJ). The anti-pLAT(Y191) ELISA kit was purchased from Cell Signaling
Technology. Lymphoprep was purchased from VWR International (West Chester, PA).
Dynabeads were purchased from Invitrogen.

3.3.3 Purification of recombinant proteins
The modified pBAD plasmid encoding the catalytic domain of HePTP (aa 44-339) in
frame with a non-cleavable 6xHis tag was a kind gift of Lutz Tautz(Mustelin, Tautz et al.
2005). cDNA fragments encoding the catalytic domains of LYP (aa 2-309) and PTP-
PEST (aa 2-323, PEST) were cloned between the BamH1 and the Xho1 sites of the
pET28a plasmid (EMD, Gibbstown, NJ) in frame with a cleavable N-terminal 6xHis-tag.
Recombinant proteins were purified from lysates of IPTG-induced E. coli BL21 cells by
affinity chromatography on Ni-nitrilotriacetic acid columns (Qiagen, Germantown, MD).
6xHis-LYP, -PTP-PEST and -HePTP were eluted using 250 mM imidazole. LMPTP-A
was cloned between the BamHI and the XbaI sites of the pEGST vector(Kholod and
Mustelin 2001), which allows expression of recombinant proteins in fusion with an N-
59
terminal GST under control of the T7 promoter. The enzyme was expressed in BL21 E.
coli cells and isolated from lysates of IPTG-induced bacteria by single-step affinity
chromatography on glutathione-sepharose (GE-Healthcare Bio-Sciences) by elution with
a glutathione solution.

3.3.4 Chemical library screening for LYP inhibitors
A library consisting of a highly diverse set of 4,000 compounds was screened in a 96-
well format in an assay specific for phosphatase activity. Each reaction contained 300
nM LYP, 2 mM pNPP, and 40 µg/mL compound in a final volume of 60 µL of 50 mM Bis-
Tris pH 6.0 reaction buffer. The reaction mixture with the compounds was incubated for
25’ at 37 °C. The reaction was stopped by addition of 50 µL of 1 M NaOH. Compounds
with an inhibition of 50% or more were further tested at multiple concentrations. All
assays were performed in triplicate. The IC
50
values were then determined for each
compound from a plot of log[inhibitor concentration] versus percentage of enzyme
inhibition. Absorbance data were measured at 405 nm using a Molecular Devices Emax
Precision Microplate reader.

3.3.5 Phosphatase assays
All reactions were carried out at 37 °C in a buffer containing 50 mM Bis-Tris, pH 6.0 and
1 mM DTT, in a final volume of 50 µL. The amount of DMSO was held constant and did
not exceed 6% of the total reaction volume. The activity of LYP and PTP-PEST was
detected using a pY peptide with sequence ARLIEDNEpYTAREG, derived from the Lck
Y394 phosphorylation site. When the pY-peptide was used as a substrate, the reaction
was stopped by addition of BIOMOL GREEN
TM
(Biomol International) and the phosphate
released was detected by measuring the absorbance at 620 nm. The time of reaction
60
and amount of enzyme for all assays were optimized in order to ensure that the readings
were taken in initial rate conditions. Unless otherwise specified, the reactions were
performed with K
M
concentration of substrate (13.72 µM for the phospho-tyrosine
peptide). The phosphatase activity measured in triplicate was corrected for the non-
specific signal of identical reactions performed also in triplicate without addition of
enzyme. The IC
50
values were then determined from a plot of inhibitor concentration
versus percentage of enzyme activity. Absorbance was measured on a Perkin Elmer
1420 Multilabel Counter Victor
3
V plate reader (Perkin-Elmer, Turku, Finland) or a Tecan
Infinite M1000 (Tecan, San Jose, CA). The data were plotted using GraphPad Prism®
(GraphPad Software, La Jolla, CA).

3.3.6 Determination of K
i
an K
i
*
The LYP-catalyzed hydrolysis of the pY peptide was measured in the presence of
various fixed concentrations of inhibitor at a series of substrate concentrations ranging
from 0.0125 mM to 0.4 mM. Data was plotted using GraphPad Prism®. Kinetic
parameters were calculated by non-linear fitting of the data to the mixed model inhibition
equation. K
i
* was calculated from the equation α = K
i
*/K
i
. (Copeland 2005).

3.3.7 Cell culture, cell treatments and TCR stimulation
Jurkat T leukemia cells expressing SV-40 large T Antigen (JTAg)(Clipstone and
Crabtree 1992) were kept at logarithmic growth in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES
pH 7.3, 2.5 mg/mL D-glucose, 100 units/mL of penicillin and 100 µg/mL streptomycin.
Compounds (50 µM) or DMSO were added to cells suspended in RPMI 1640, and
incubated for 1h at 37 °C. The volume of DMSO added was held constant at less than or
61
equal to 2% of the total volume. Cells were then divided and stimulated at 37 ° or left
unstimulated. JTAg cells were stimulated with supernatants of C305 hybridoma(Weiss
and Stobo 1984) for 2’. Primary human T cells were stimulated for 1’ with 1 µg/ml anti-
CD3, 1 µg/ml anti-CD28, and 10 µg/ml rabbit anti-mouse F(ab’)2 crosslinker.
Thymocytes were stimulated by incubation with biotinylated anti-CD3 and anti-CD4
antibodies for 30’ and stimulated with a streptavidin crosslinker for 1.5’.

For Western blot analysis, cells were lysed in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5
mM EDTA pH 8.0 and 1% NP-40 (TNE), containing 10 µg/mL aprotinin and leupeptin,
10 µg/mL soybean trypsin inhibitor, 1 mM Na
3
VO
4
and 1 mM phenylmethylsulfonyl
fluoride. For analysis by ELISA, cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 1 mM Na
2
EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
beta-glycerophosphate, 1 mM Na
3
VO
4
and 1 µg/ml leupeptin. Lysates were clarified by
centrifugation at 13,200 rpm for 20’.

3.3.8 Primary cell isolation
Thymocytes were isolated from the homogenized thymi of 4-6 week old C57BL/6
(PTPN22+/+) mice (Taconic Farms, Inc., Hudson, NY) or PTPN22-/- mice (Genentech,
Inc., South San Francisco, CA) after depletion of red blood cells following standard
procedures. Primary human T cells were isolated by Lymphoprep gradient centrifugation
followed by depletion of B cells and monocytes by anti-CD19 and anti-CD14 Dynabeads.

3.3.9 Phospho-flow cytometry
Phospho-flow cytometry was performed on JTAg cells following published protocols (BD
Biosciences). Cells were treated with 50 µM compound 4 or DMSO, stimulated with
62
C305 for 2’ or left unstimulated, and fixed immediately with BD Cytofix buffer. Cells were
permeabilized using BD Phosflow III and stained with PE-conjugated anti-pSLP76(Y128)
antibody. Cell fluorescence was assessed on a Cytomics FC500 cytometer (Beckman
Coulter) at the USC Immune Monitoring Core. Data analysis and graph preparation were
performed using FlowJo software (Tree Star, Inc., Ashland, OR). Differences between
distributions were calculated by Overton subtraction(Overton 1988). The sampling error
in C305-stimulated JTAg cells was evaluated by measuring the average±SD Overton
subtraction % of three independent replicates and was 2.5±0.6% for the anti-pSLP76
antibody.

3.3.10 T cell activation assay
For induction of CD69, cells were stimulated with 1 µg/mL anti-CD3 and anti-CD28 Abs
for 24 hours at 37 ° in the presence of compound 4 or DMSO alone. Cells were then
stained with anti-CD69-PerCP-Cy5.5 and either anti-CD4-FITC or anti-CD8-FITC
conjugated antibodies. Gates were set on CD4
+
and CD8
+
cells and the percentages of
activated CD69
hi
cells were calculated. Samples were acquired on a FACSCanto II (BD
Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR).

3.4 Results
3.4.1 Enzyme-based screening and counter-screening
We initially screened a library of drug-like small-molecule compounds against LYP, using
pNPP as a substrate. The library consisted of 4000 compounds, which had been
selected from the Asinex (Winston-Salem, NC) collection based upon high predicted cell
permeability features. A total of 137 compounds exhibited > 50% inhibition at a
concentration of 40 µg/mL and were further tested at different concentrations to calculate
63
IC
50
values. The LYP inhibitory activity of these 137 compounds ranged from 11-100 µM.
Next, we excluded compounds that, at visual inspection, suggested evidence of
oxidation-mediated inhibition. Twelve of the remaining compounds showed IC
50
values
less than or equal to 30 µM, and their structures are shown in Table 3.1. These
compounds were tested for selectivity against 3 additional PTPs, including PTP-PEST
(the PTP most closely related to LYP), HePTP and LMPTP-A(Alonso, Sasin et al. 2004).
As shown in Table 3.2, half of the compounds (compounds 1, 2, 4, 5, 6 and 7) showed
some selectivity for LYP over PTP-PEST. Of these compounds, four (compounds 1, 2, 4
and 5) showed no selectivity between LYP and HePTP, while compound 6 showed
around two times selectivity for LYP. Compound 7 showed higher selectivity for HePTP
over LYP, and was excluded from further consideration. The remaining top five
compounds (compounds 1, 2, 4, 5 and 6) showed little inhibitory activity on LMPTP-A,
which is the least structurally related enzyme to LYP.

64
Table 3.1. Structures of top LYP small molecule inhibitors. Compounds are ranked by decreasing potency
(IC 50 in LYP inhibition assays).

# Structure # Structure
1 7

2 8

3 9

4 10

5 11

6 12

65
Table 3.2. Potency and selectivity of top LYP small molecule inhibitors. Performed by Roza Kazemi and Lei
Zhao.
LYP PTP-PEST HePTP LMPTP-A
Compound IC
50
(µM) IC
50
(µM) IC
50
(µM) IC
50
(µM)
1 11.2 ± 0.5 20±3 9±0 >90
2 11.3 35±5 10±2 71.9
3 19.8 ± 1 21±1 8±2 45.3
4 20.6 ± 2 >40 20±2 >90
5 22 >40 18±3 >90
6 22.0 ± 2 39±1 >40 >90
7 23.3 ± 2.8 36±2 12±0.5 >90
8 26.6 ± 3 27.5±0.5 20±2 53.3
9 27.2 ± 3 28±4 22 >90
10 28.1 ± 2 16±4 16±3 >90
11 29.5 ± 3.3 14±1 8±2.5 >90
12 30.5 ± 0.5 18±5 15±2 60.9

Next, in preparation for cell-based assays, we carried out a secondary screen to test the
inhibitory activity of the top five compounds on LYP using a 14-amino acid peptide,
ARLIEDNEpYTAREG, derived from the Y394 phosphorylation site of Lck, which more
closely resembles the physiological substrate of the phosphatase. Four of the
compounds showed IC
50
values around 10 µM (IC
50
was 6.2 µM on compound 1, 5.4 µM
on compound 2, 13.9 µM on compound 4, and 11.0 µM on compound 6), while
compound 5 had an IC
50
value > 80 µM, and was not pursued further.

3.4.2 Effect of inhibitors on TCR signaling
Next, we tested the top four inhibitors for their ability to inhibit LYP in T cells. First, we
tested the effects of the four compounds on the early stages of TCR signaling in the
66
Jurkat T Antigen (JTAg) human T cell line(Clipstone and Crabtree 1992). As a read-out
of PTP activity, we analyzed the phosphorylation of Lck, a protein tyrosine kinase (PTK)
involved in the initial stages of TCR signaling, at its positive regulatory Y394 residue. Lck
is a well-known physiological substrate of the PTPs CD45 and LYP through its two major
phosphorylation sites at Y505 (a negative regulator of Lck activation) and Y394 (a
positive regulator of Lck activation), respectively (Mustelin, Alonso et al. 2004; Wu,
Katrekar et al. 2006). Following incubation with LYP inhibitors (50 µM of compounds 1,
2, 4, and 6) for 1 h, we stimulated JTAg cells with C305 antibody(Weiss and Stobo 1984)
for 2’ at 37 °C. Lysates of these cells were probed with anti-pSrc(Y416), an antibody
which selectively recognizes the phosphorylated Y416 in Src, and in T cell lysates cross-
reacts with the equivalent pY394 site of Lck. As shown in Fig. 3.1A, among the four
inhibitors tested, only compound 4 increased TCR-induced phosphorylation of Lck at
Y394.

To directly test if the increase in Y394 phosphorylation was due to inhibition of LYP, we
analyzed the effect of compound 4 on thymocytes of Ptpn22-/- knockout mice compared
to normal Ptpn22+/+. As seen in Fig. 3.1B, compound 4 caused an increase in Lck
phosphorylation at Y394 in wild type (Ptpn22+/+) thymocytes, but had no effect on
phosphorylation of Lck at the same site in Ptpn22-/- thymocytes, suggesting that
compound 4 preferentially inhibits PEP (the mouse homolog of LYP) over other
phosphatases.

67
Figure 3.1. Intracellular inhibition of LYP by compound 4. (A) Top panels, anti-pSrc(Y416) immunoblots
of lysates of Jurkat TAg cells treated with 50 µM of the top four compounds in Table 1 (lanes 3 and 4 of each
panel) or untreated (lanes 1 and 2 of each panel) and either left unstimulated (lanes 1 and 3 of each panel)
or stimulated (lanes 2 and 4 of each panel) with C305 for 2’. Bottom panels, anti-Lck blot of same samples.
Treatment with compound 4 caused identical effects in three independent experiments. Performed by Divya
Krishnamurthy. (B) Top panels, anti-pSrc(Y416) immunoblots of lysates of thymocytes from PTPN22 -/- (left
panels) or PTPN22 +/+ (right panels) mice treated with 50 µM of compound 4 (lanes 3 and 4 in each panel)
or untreated (lanes 1 and 2 in each panel) and either left unstimulated (lanes 1 and 3 in each panel) or
stimulated (lanes 2 and 4 in each panel) with biotinylated anti-CD3 and anti-CD4, followed by cross-linking
with streptavidin for 1.5’. Bottom panels, anti-Lck blot of same samples. Figure is representative of two
independent experiments. Arrows indicate the position of Lck in each panel. Performed by Divya
Krishnamurthy.

B.
68

We proceeded to evaluate the effects of compound 4 on downstream signaling events
such as the activation of ZAP70, and on the Ras-Raf-MAPK pathway (ERK1/2) in JTAg
cells. In agreement with the results observed with Lck (replicated in Fig. 3.2A, top
panel), compound 4 increased TCR-stimulated phosphorylation of ZAP70, as seen from
the anti-pZAP(Y319) blot (Fig. 3.2A, middle panel). In addition, compound 4 also
increased ERK1/2 phosphorylation, as evidenced by the anti-pERK1/2 blot (Fig. 3.2A,
lower panel). We also assessed the effect of the inhibitor on TCR-induced
phosphorylation of SLP76 at the single-cell level by phospho-specific flow cytometry
(Fig. 3.2B). SLP76 is an early mediator of TCR signaling downstream of Lck and LYP.
Compared to the cells treated with DMSO alone, as seen in Fig. 3.2B, cells treated with
compound 4 showed higher levels of TCR-induced SLP76 phosphorylation.

To extend our observation on compound 4 to primary human cells, we tested its effect
on TCR-induced phosphorylation of LAT, another early mediator of TCR signaling
downstream of Lck and LYP. Fig. 3.2C shows that treatment of primary human T cells
with compound 4 caused an increase in TCR-induced LAT phosphorylation.

We next assessed whether compound 4 could increase downstream T cell activation.
Table 3.3 shows that treatment with compound 4 caused a dose-response increase in
TCR-induced expression of CD69, a marker of activated T cells(Nurmi, Autero et al.
2007), in both CD4
+
and CD8
+
primary mouse splenocytes.

Taken together, all these results demonstrate that compound 4 is efficient in boosting T
cell activation by suppressing the function of LYP. Overall, these data suggest that
69
compound 4 is a good candidate inhibitor for testing in animal models of autoimmunity
pending further optimization of potency and selectivity.
70
Figure 3.2. Treatment of T cells with compound 4 increases TCR signaling downstream Lck. (A)
Jurkat TAg cells were treated with 50 µM compound 4 (lanes 3 and 4 in each panel) or left untreated (lanes
1 and 2 in each panel) and were either left unstimulated (lanes 1 and 3 in each panel) or stimulated (lanes 2
and 4 in each panel) with C305 for 2’. Panels show blots of total lysates with the following antibodies: top
panel, anti-pSrc(Y416); second from top, control anti-Lck; third from top, anti-pZAP70(Y319); third from
bottom, control anti-ZAP70; second from bottom, anti-pERK; bottom panel, control anti-ERK. Performed by
Divya Krishnamurthy. (B) Single-cell study of TCR-induced phosphorylation of SLP-76 by phospho-flow
cytometry. Jurkat TAg cells were treated with 50 µM compound 4 (continuous line and black graphs) or left
untreated (dashed-line and grey graphs), and either left unstimulated (white graphs) or stimulated with C305
for 2’ (grey and black graphs). Cells were fixed and stained with a PE-conjugated anti-pSLP76(Y128)
antibody. Graphs show expression of pSLP76 levels as detected by flow cytometry after gating on high
forward and high side scatter (=cells with high SLP76 phosphorylation) cells. The statistical significance of
the difference between compound 4-treated and untreated cells was calculated by Overton subtraction
(Overton 1988). Data are representative of two experiments with identical results. Performed by Divya
Krishnamurthy. (C) Primary human T cells were treated with 50 µM compound 4 (black histograms) or
DMSO (gray histograms) and were either left unstimulated or stimulated with anti-CD3, anti-CD28 and
crosslinked for 1’. Graph shows relative levels of pLAT(Y191) as assessed by ELISA.

71
Figure 3.2: Continued

A.
B.
C.
72
Table 3.3. Compound 4 causes increased T cell activation.

3.4.3 Mechanism of inhibition by compound 4
In prevision to the use of compound 4 or analogs in animal experimentation, we carried
out kinetic analysis of its possible mechanism of inhibition using the above-mentioned
pY peptide as a substrate. As shown in Fig. 3.3A, compound 4 shows a mixed
competitive and non-competitive inhibition mechanism, with a K
i
of 8.5 ± 6.4 µM, and a
K
i
’ of 19.7 ± 6.8 µM. We compared the mechanism of inhibition of compound 4 with the
competitive inhibitor reported by the Zhang group, I-C11(Yu, Sun et al. 2007). The
comparison of the two compounds in Fig. 3.3 revealed that I-C11 displayed a
competitive phenotype, causing an increase in K
M
with increasing inhibitor concentration,
without decreasing V
Max
. In contrast, increasing concentrations of compound 4 caused a
decrease in V
Max
with only a small increase in K
M
at high concentrations of inhibitor,
which is consistent with at least a partial non-competitive mechanism(Copeland 2005).

To confirm the mechanism of action of compound 4, we measured the IC
50
on LYP with
increasing substrate concentration. Competitive inhibitors show decreased potency with
increasing substrate concentration, while noncompetitive inhibitors have IC
50
values that
are unaffected by the substrate concentration(Copeland 2005). Fig. 3.3G shows that
Sample % CD69
hi
Cells
CD4
+
DMSO 29.2
CD4
+
50 µM Comp 4 36.8
CD4
+
100 µM Comp 4 38.9
CD8
+
DMSO 5.34
CD8
+
50 µM Comp 4 12.1
CD8
+
100 µM Comp 4 13.7
73
increasing the concentration of pY peptide led to an increase in the IC
50
of I-C11,
consistent with competitive inhibition, but did not affect the IC
50
of compound 4.
Figure 3.3. Mechanism of inhibition of compound 4. (A) Activity of 25 nM LYP on the pY peptide in the
presence of increasing concentrations of compound 4. Points are the average ±SD of the activity of LYP
measured in triplicate plotted vs substrate concentration. Lines are fits of data to the Michaelis-Menten
equation. (B-C) Plot of concentration of compound 4 versus V Max (B) or K M (C) of the data shown in (A). (D)
Activity of 25 nM LYP on the pY peptide in the presence of increasing concentrations of I-C11. Points are
the average ±SD of the activity of LYP measured in triplicate plotted vs substrate concentration. Lines are
fits of data to the Michaelis-Menten equation. (E-F) Plot of concentration of I-C11 versus V Max (E) or K M (F)
of the data shown in (D). Ki and α parameters were calculated for the data shown in (A) and (C) by fitting of
the data to the mixed model inhibition equation. (G) Plots of IC 50 of compound 4 (red diamonds) or I-C11
(blue diamonds) on LYP on the pY peptide versus substrate concentration/K M. Points show the IC50 ± 95%
confidence intervals.

A.
B.
C.
74
Figure 3.3: Continued

D.
F.
E.
G.
75
3.4.4 Analogs of compound 4
We next attempted to determine if we could identify analogs of compound 4 with
increased potency on LYP or selectivity compared to PTP-PEST. As shown in Fig. 3.4,
compound 4 has three domains which can be explored with substitutions. On Domain 1,
we compared the activity and selectivity of compound 4 with four analogs with
substitutions on the tetrazole ring on both LYP and PTP-PEST using the pY peptide as a
substrate. Table 3.4 shows that three of these analogs displayed both increased
potency and selectivity compared to compound 4. On Domain 2, we tested a variety of
substitutions and found three analogs with increased potency and selectivity (Table 3.5).
On Domain 3, we tested six analogs with alkyl or methoxy substitutions on the quinolin-
2(1H)-one group, and found two analogs with alkyl substitutions which increased both
potency and selectivity (Table 3.6).

Compound 4 seems to be a good candidate for further optimization to obtain lead(s) with
sufficient potency and selectivity to enable future studies of efficacy in animal models of
autoimmunity.

Figure 3.4. Domains of compound 4.

76

Table 3.4. Potency and selectivity of substitutions of Domain 1.

R1 Substitutions LYP IC
50

Selectivity
Over PTP-
PEST

13.85 +/- 0.10 1.20
A

6.07 +/- 5.58 1.61
B

10.27 +/- 0.33 1.53
C

11.18 +/- 3.46 1.36
D

21.24 +/- 5.41 1.33

77

Table 3.5. Potency and selectivity of substitutions of Domain 2.

R2 Substitutions LYP IC
50

Selectivity
Over PTP-
PEST

13.85 1.20
A

5.29 +/- 1.04 1.05
B

6.30 +/- 0.30 1.38
C

9.19 +/- 1.79 1.85
D

13.03 +/- 1.23 1.16

78

Table 3.5: Continued

E

13.30 +/- 1.20 1.48
F

17.12 +/- 2.48 1.55
G

20.58 +/- 2.16 1.52
H

25.09 +/- 1.44 0.92
I

30.06 +/- 3.65 1.28
J

30.54 +/- 5.81 1.28
K

31.50 +/- 9.78 1.27
L

30.85 +/- 0.48 2.10
79

Table 3.5: Continued

M

30.55 +/- 0.66 1.16
N

30.71 +/- 0.44 0.89

80

Table 3.6. Potency aMnd selectivity of substitutions of Domain 3.

R3 Substitutions LYP IC
50

Selectivity
Over PTP-
PEST

13.85 1.20
A

11.11 +/- 0.35 1.44
B

12.12 +/- 1.07 1.33
C

15.17 +/- 0.75 1.43
D

15.75 +/- 1.58 1.34
E

18.16 +/- 0.69 1.74
F

20.40 +/- 3.47 1.53

81
3.5 Discussion
The recent success of anti-CD3 therapy in clinical trials for T1D and other autoimmune
diseases suggests that acute positive modulation of T cell signaling might be beneficial
for treatment of autoimmunity, most likely through induction of immunosuppressive
regulatory T cells(Chatenoud 2006). Beside direct engagement of the TCR/CD3,
increased T cell activation can also in principle be achieved by inhibition of negative
modulators of signaling. Among those, the protein tyrosine phosphatase LYP is
considered a high priority target, due to the mentioned genetic and functional evidence
in humans showing that gain-of-function of LYP causes autoimmunity(Begovich, Carlton
et al. 2004; Bottini, Musumeci et al. 2004). It has been proposed that a small molecule
inhibitor of LYP would have preventative and therapeutic efficacy in patients with a wide
range of autoimmune disease, especially in carriers of the hyperactive W620
variant(Vang, Congia et al. 2005; Yu, Sun et al. 2007). Deletion of the phosphatase in
the mouse results in a phenotype of exclusive increased TCR signaling without effects
on other signaling pathways or appearance of evident pathology(Hasegawa, Martin et al.
2004). This suggests that pharmacological inhibition of LYP is unlikely to result in severe
side effects. In addition to their possible value as therapeutic agents, small molecule
inhibitors of LYP would also help understanding the biology of this important
autoimmunity gene, by providing a complementary approach to germline knockout of the
phosphatase in mice.

Tyrosine phosphatases are emerging as promising drug targets for cancer, metabolic
and inflammatory diseases(Dawson, Xia et al. 2008; Heneberg 2009). On account of the
highly charged active site, many PTP inhibitors are phospho-tyrosine mimics(Lee, Zhang
et al. 2003; Yu, Sun et al. 2007; Xie, Liu et al. 2008), which sometimes constitutes a
82
limitation to their cell-permeability(Blaskovich 2009). Another challenge of PTP inhibitor
discovery is the high degree of conservation of the active site among classical PTPs,
which is an obstacle to the identification of highly specific inhibitors(Blaskovich 2009).
So far, only a few small molecule inhibitors of LYP have been described (Yu, Sun et al.
2007; Krishnamurthy, Karver et al. 2008; Xie, Liu et al. 2008; Wu, Bottini et al. 2009).
These compounds have been identified using novel approaches, including bidentate
chemistry(Zhang 2003) and in silico comparative screening of multiple active site
conformations(Wu, Bottini et al. 2009), which helped to increase their potency and
specificity profile. However, all the competitive LYP inhibitors identified so far have had
phospho-mimetic features.

In our efforts to obtain LYP inhibitors, we sought to focus on cell-permeability. We
previously reported the identification of cell-permeable LYP inhibitors among chemically
synthesized analogs of Auranofin, a gold-based compound with known efficacy in
autoimmune diseases(Krishnamurthy, Karver et al. 2008). The goal of the present study
was to identify cell-permeable LYP inhibitors by screening a library of small molecules
which had been selected to maximize drug-like properties. This approach resulted in the
identification of compounds that inhibit LYP with IC
50
ranging from 11-30 µM.
Interestingly, none of the scaffolds reported in this manuscript seem to be phospho-
mimetic.

Upon testing our top four inhibitors in T cells, only one compound (compound 4) was
able to significantly increase TCR-induced Lck, ZAP70, SLP76 and ERK
phosphorylation. Key evidence that the effects of compound 4 on TCR signaling are due
to selective inhibition of LYP was obtained by using primary cells from PTPN22+/+ and
83
PTPN22-/- mice. A possible explanation for the lack of activity of compounds 1, 2, and 6
in cell-based assays could be their concurrent inhibition of CD45, a positive regulator of
Lck activation. To exclude this possibility, we assessed whether the top four compounds
would inhibit CD45 in vitro. We found that compound 2 was able to inhibit CD45 with an
IC
50
of 11 µM, while the other compounds were poor CD45 inhibitors (IC
50
were 87 and
90 µM for compounds 4 and 1, respectively, and > 90 µM for compound 6). However, we
were unable to detect increased phosphorylation of Lck at Y505 in JTAg cells treated
with compound 2 (data not shown). Thus at the moment, despite the non-phospho-
mimetic features of compounds 1, 2, and 6, lack of cell-permeability likely underlies their
poor performance in cell-based assays.

In conclusion, by screening a library of selected drug-like compounds, we were able to
identify a cell-permeable inhibitor of LYP. Although only a minority of our top compounds
were active in cell-based assays, our results suggest that careful selection of the
chemical library might lead to an increased chance to identify cell-permeable inhibitors of
PTPs.

84
Chapter 4: Single-cell assay of tyrosine phosphatase activity using fluorogenic
pCAP peptides
4.1 Abstract
Several protein tyrosine phosphatases (PTPs) are candidate drug targets, however
development of drug-like small molecule inhibitors of PTPs remains a challenging task.
Cell-based assays of PTP activity are desirable in order to identify candidate PTP
inhibitors with favorable cell permeability and toxicity properties. Here we developed a
method to monitor tyrosine phosphatase activity at the single-cell level, by taking
advantage of the fluorogenic properties of phosphorylated coumarin amino propionic
acid (pCAP), an amino acid analog of phosphotyrosine, which can be easily incorporated
into peptides. Once delivered into cells, pCAP peptides are exclusively
dephosphorylated by PTPs and the resulting cell fluorescence can be easily monitored
in high content imaging. Our assay can be applied to screening of PTP inhibitor libraries
to avoid/eliminate hits with poor cell permeability or other unfavorable properties.

4.2 Introduction
Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction
pathways controlling cell metabolism, growth and differentiation(Alonso, Sasin et al.
2004). Several PTPs are considered excellent drug targets for common and debilitating
human diseases, including cancer, diabetes, arthritis, and infectious diseases(Tautz,
Pellecchia et al. 2006; Jiang and Zhang 2008; Heneberg 2009). To mention just one
example, inhibitors of the tyrosine phosphatase PTP-1B, a well-known regulator of
insulin signaling, are currently sought after for therapy of type 2 diabetes and of the
metabolic complications of obesity(Zhang and Lee 2003; Zhang and Zhang 2007).
However, development of small molecule inhibitors of PTPs can be a challenging task.
85
Due to the highly conserved, positively charged character of the active site of PTPs, the
most potent hits identified by chemical library screening using enzyme-based assays
commonly show low specificity and poor cell-permeability(Blaskovich 2009). Especially
poor cell-permeability of PTP candidate inhibitors (which usually carry a negative
charge) has been a serious limitation, which has been difficult to overcome in later
stages of drug development. Novel cell-based assays able to directly monitor
intracellular PTP activity and suited to high-throughput screening would be desirable to
speed up development of cell-permeable PTP inhibitors.

Several colorimetric and fluorogenic assays based on the dephosphorylation of non-
peptidic probes are available for use in enzyme-based screening for small molecule
inhibitors of PTPs(Montalibet, Skorey et al. 2005). For example, para-nitrophenyl
phosphate (pNPP) is readily dephosphorylated by most PTPs, yielding the yellow-
colored para-nitrophenol compound. Coumarin-based compounds like 4-
methylumbelliferyl phosphate (MUP) and its fluorinated derivative 6,8-difluoro-4-
methylumbelliferyl phosphate (DiFMUP) are also commonly used substrates for
screening of PTP inhibitors(Montalibet, Skorey et al. 2005; Lavis and Raines 2008).
Dephosphorylation of these compounds by PTPs yields highly fluorescent derivatives.
These assays are very robust and routinely used in primary screening of libraries for
PTP inhibitors.

Previously, we reported the development of an amino acid version of MUP,
phosphorylated coumarin amino propionic acid (pCAP, see Fig. 4.1A), which can be
incorporated into peptides using routine FMOC chemistry(Mitra and Barrios 2005). We
showed that pCAP peptides are easily dephosphorylated into CAP (see Fig. 4.1A) by
86
PTPs(Mitra and Barrios 2005), and can be developed into fluorogenic direct enzyme-
based assays for primary screening of PTP inhibitors(Karver, Krishnamurthy et al. 2009).
We also generated fluorinated derivatives of pCAP and showed that they can be used in
PTP assays as well(Mitra and Barrios 2007). Enzyme-based assays based on pCAP
peptides have similar characteristics of robustness as those based on MUP and
DiFMUP(Karver, Krishnamurthy et al. 2009). However, the peptidic nature of pCAP-
based probes confers some unique advantages for primary screening of libraries. For
example, pCAP peptides offer an interaction surface closer to the physiological
substrate, and their sequence can be optimized by peptide library screening(Mitra and
Barrios 2008).

While much effort is going into developing improved in vitro enzyme-based PTP assays,
very few reports have dealt with cell-based assays for directly monitoring intracellular
tyrosine phosphatase activity. All of the assays so far are based on measuring the
dephosphorylation of non-peptidic probes incubated with cells at the population level
using a plate-reader. For example, the Kennedy group monitored dephosphorylation of
pNPP by insect cells infected with baculoviruses encoding PTP-1B or CD45(Cromlish,
Payette et al. 1999), while Peters et al. reported an assay based on the
dephosphorylation of DiFMUP by 293 cells stably overexpressing PTP-1B(Peters, Davis
et al. 2003). Although the use of a plate-reader ensures considerable throughput, signal
detection at the population level might introduce false positives due to cell-death
following virus infection and/or compound toxicity, followed by extracellular
dephosphorylation of the probe. Also, small non-peptidic probes may have poor
permeability properties and are not actively concentrated in cells, thus decreasing the
available signal at the single-cell level. Some of these probes might also be
87
dephosphorylated by other enzyme families (for example DiFMUP is also
dephosphorylated by serine-threonine phosphatases(Pastula, Johnson et al. 2003;
Welte, Baringhaus et al. 2005)) thus increasing the noise of the assay.

In this report, we describe the development and optimization of an assay to monitor
tyrosine phosphatase activity at the single-cell level, by using cell-permeable pCAP
peptides. Once delivered into the cells, the pCAP peptides are exclusively
dephosphorylated by PTPs. The intracellular spontaneous accumulation of pCAP
peptides was achieved by using an optimized cell-permeable tag. The
dephosphorylation of cell-permeable peptides by PTPs generated a signal that can be
easily monitored by flow cytometry and fluorescence microscopy. Our assay is suited to
high-content screening platforms, and can be applied in primary or secondary screening
of chemical libraries to avoid/eliminate candidate PTP inhibitors with poor cell-
permeability, high cellular toxicity, and other unfavorable properties.

4.3 Experimental Procedures
4.3.1 Reagents
Sodium orthovanadate and protease inhibitors were purchased from Sigma (St. Louis,
MO). AlexaFluor-555 dextran, Sytox Orange, and CellMask Deep Red were purchased
from Invitrogen (Carlsbad, CA). Recombinant PP-1 was purchased from
Upstate/Millipore (Billerica, MA). Recombinant PP-2A and PP-2B were purchased from
Calbiochem/EMD (Gibbstown, NJ). Recombinant YopH and SHP-1 were purchased
from Biomol International (Plymouth Meeting, PA).

88
Fmoc protected amino acids, Rink Amide AM resin, and HCTU were purchased from
Novabiochem. N,N-Dimethylformamide (DMF), trifluoroacetic acid (TFA), and 1,8-
Diazabicyclo[5.4.0]undec7-ene (DBU) were purchased from Alfa Aesar.
Diisopropylcarbodiimide (DICI) and triisopropylsilane (TIS) were purchased from Acros
Organics. Diisopropylethylamine (DIPEA), trimethylsilyliodide (TMSI), dichlormethane
(DCM), methanol, and acetic acid were purchased from Fisher Scientific. 1-
Hydroxybenzotriazole (HOBT) was purchased from ChemImpex (Wood Dale, IL). All
reagents were used without further purification. The non-natural amino acid pCAP was
synthesized as previously reported(Mitra and Barrios 2005). Peptide synthesis vessels
were comprised of a fritted column purchased from Whatman Inc., teflon stopcock and
cap purchased from Applied Separations. Peptides were purified on a Varian ProStar
210 preparative-scale HPLC and characterized by MALDI-TOF on a DE-STR
(PerSeptive Biosystems/ ABI) instrument by the University of Utah Mass Spectrometry
and Proteomics Core Facility using α-cyano-4-hydroxycinnamic acid as matrix.

4.3.2 Synthesis of Ac-EDNE-X-TARE-NH
2
, where X = pCAP, CAP, and Et
2
-pCAP
Rink Amide AM resin (50 mg, 0.035 mmol) was pre-swelled in 1 mL DMF in the peptide
synthesis vessel and shaken for 30 min. After draining, 1 mL of 2% DBU in DMF was
added and allowed to shake for 30 min. The solution was drained and another 1 mL of
2% DBU was added and shaken for 30 min more. The fully deprotected resin was then
washed with DMF (3 x 1 mL). The desired amino acid (3 equiv) was dissolved in 1 mL
DMF along with HOBT and DICI (0.105 mmol, 3 equiv each) and allowed to sit for 10
min. The mixture was added to the rinsed resin and shaken vigorously for 3 h. After
coupling, the solution was drained and rinsed with DMF (3 x 1 mL) and deprotected with
1 mL 2% DBU (2 x 30 min). The next amino acid was then coupled and the cycle was
89
repeated until each amino acid was added. The amino acid N-terminal to pCAP was
coupled twice to ensure complete reaction. The first coupling was performed using the
conditions described above, rinsed with DMF (3 x 1 mL) and coupled a second time
using 5 equiv of amino acid, HCTU (0.175 mmol, 5 equiv) and DIPEA (0.35 mmol, 10
equiv) and shaken for 1.5 h. After deprotection of the final amino acid with 2% DBU, the
N-terminus was acetylated using acetic acid/HCTU/DIPEA (5 equiv, 5 equiv, 10 equiv,
respectively) for 1.5 h. The resin was then washed with DMF (3 x 1 mL) and DCM (3 x 1
mL) and dried under vacuum. All pCAP containing peptides were deprotected with a 1 M
solution of TMSI in DCM for 1 h. The resin was then washed with DCM (3 x 1 mL) and
methanol (3 x 1 mL) and dried under vacuum. The peptides were then cleaved from the
resin using a cleavage cocktail containing 95% TFA, 2.5% water, and 2.5% TIS for 4 h.
The peptides were collected under vacuum. The resin was rinsed with 1 mL TFA and 10
mL water. The collected solution was diluted with approx 40 mL water and lyophilized.
The peptides were then purified on HPLC using water, acetonitrile and TFA buffers, and
lyophilized again. The sequence of the pCAP, CAP, and Et
2
-pCAP peptides were EDNE-
X-TARE, where X = pCAP, CAP, and Et
2
-pCAP, respectively. The peptides were
characterized using MALDI-TOF MS. MALDI TOF MS for C
50
H
71
N
14
O
26
P (EDNE-pCAP-
TARE): [M+H
+
] 1315 calc., 1315 found. MALDI TOF MS for C
50
H
70
N
14
O
23
(EDNE-CAP-
TARE): [M+H
+
] 1235 calc., 1236 found. MALDI TOF MS for C
54
H
79
N
14
O
26
P (EDNE-
pCAP(Et)
2
-TARE): [M+H
+
] 1371 calc., 1373 found.

4.3.3 Synthesis of C
14
-B-R
7
-EDNE-X-TARE-NH
2
(where X = CAP and pCAP)
The peptide was synthesized following the same procedure as for the Ac-EDNE-pCAP-
TARE-NH
2
using 75 mg of Rink-amide resin and 3 equiv of each amino acid. After the
removal of the Fmoc-protecting group from the N-terminus glutamate, the arginine
90
residues using 3 equiv of Fmoc-Arg(Pbf)-OH (103.6 mg, 0.25 mmol) with 3 equiv DICI
(25 µL, 0.25 mmol) and 3 equiv HOBt (21.6 mg, 0.25 mmol) in 1 mL of DMF were added
consequentially. After the seventh arginine residue, β-Ala (B) was attached using the
same coupling conditions. The tetradecanoic fatty acid tail was added using two
consecutive coupling conditions. Myristoyl chloride (76.7 µl, 0.28 mmol) was dissolved in
3 mL of dry DCM. To this solution 0.6 mL of DIPEA was added and mixture was added
to the resin immediately and reacted for 2 h under nitrogen atmosphere. After the
reaction was complete the resin was drained and washed with DCM. A solution of 5
equiv tetradecanoic acid (81 mg, 0.35 mmol) in 1 mL of DCM and 1 mL of DMF, with 4.9
equiv of HCTU (143.9 mg, 0.35 mmol) and 10 equiv of DIPEA (122.8 µl, 0.7 mmol) was
added to the resin and the reaction proceeded for 2 h. After completion the resin was
washed with DMF (3 × 1mL) and DCM (3 × 1mL) and dried completely. To cleave the
peptide from the resin 1 mL of TFA solution (950 µl of TFA, 25 µl of water, 25 µl of TIS)
was added to the dry resin containing the peptide and stirred overnight. The crude
peptide was collected and water was added to 30 mL. To remove the TFA the mixture
was lyophilized. The crude peptide was purified on HPLC using water, acetonitrile and
TFA buffers. The peptides were characterized using MALDI TOF MS. For C
14
-B-R
7
-
EDNE-pCAP-TARE-NH
2
, C
107
H
184
N
43
O
34
P: [M+H
+
] 2646 calc., 2630 found. For C
14
-B-R
7
-
EDNE-CAP-TARE-NH
2
, C
107
H
183
N
43
O
31
: [M+H
+
] 2567 calc., 2569 found.

4.3.4 In vitro kinase assays
For detection of PTK activity, 0.4 mM substrate was incubated with enzyme in 20 mM
Hepes pH 7.4, 5 mM MnCl
2
, 20 mM MgCl
2
, 0.1% Triton-X, 0.1% NP40, 1 mM ATP and 5
mM DTT at 37°C. The reactions were monitored in triplicate by continuously measuring
the fluorescence ( λ
exc
= 340 nm and λ
em
= 460 nm).
91
4.3.5 In vitro phosphatase assays
For detection of PTP activity, substrates were incubated with enzyme in 50 mM Bis-Tris
pH 6.0 with 1 mM DTT. Assays of PP-1 activity were conducted in 50 mM Tris-HCl pH
7.0, 5 mM DTT, 2 mM MnCl
2
, 0.1 mM EDTA, and 0.2 mg/mL BSA. Assays of PP-2A
activity were conducted in 50 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.9 mg/mL BSA,
0.09% β-mercaptoethanol, and 1 mM MnCl
2
. Assays of PP-2B activity were conducted
in 20 mM Tris-HCl pH 7.5, 10 mM MgCl
2
, 0.1 mM CaCl
2
, 1 mg/mL BSA, and 1 mM DTT.
All reactions were carried out at 37°C. The reactions were monitored in triplicate by
continuously measuring the increase in fluorescence ( λ
exc
= 340 nm and λ
em
= 460 nm for
pCAP substrates and λ
exc
= 355 nm and λ
em
= 460 nm for DiFMUP substrates). The
activity of serine/threonine phosphatases was normalized using DiFMUP as substrate
and equal units were used for the pCAP assays.

4.3.6 Microinjection of sea urchin oocytes
Eggs were spawned from an adult sea urchin female. Jelly coat was removed by
treatment with an acidic seawater (pH 4.0) for 1 min and stored at 12°C until ready for
injection. Sperm was collected from adult males and stored at 4°C till use. Eggs were
fertilized in the presence of 150 mM PABA seawater (paraminobenzoic acid seawater)
and injected using a picospritzer as described over a Zeiss inverted microscope(Damle,
Hanser et al. 2006). To correct for cell/injection volumes, and as an injection marker,
fixed amounts of rhodamine were co-injected with the peptides.

4.3.7 Cell culture, stimulation and transfections
Jurkat E6.1 cells were grown in RPMI 1640 medium supplemented with 10% FBS, 1 mM
sodium pyruvate, 10 mM HEPES pH 7.3, 2.5 mg/mL D-glucose, 100 units/mL of
92
penicillin and 100 µg/mL streptomycin. RAW 264.7 and COS-7 cells were grown in
DMEM supplemented with 10% FBS, 100 units/mL of penicillin and 100 µg/mL
streptomycin. All cells were maintained at logarithmic growth. For treatment of Jurkat
with cell-permeable peptides, peptides were added at the appropriate concentration to
cells in RPMI with 0.5% FBS and cells were incubated at 37 °C with 5% CO
2
for the time
indicated in the figures. For treatment of COS-7 or RAW 264.7 with cell-permeable
peptides, cells were plated and allowed to adhere overnight in culture medium. Cells
were then washed and incubated with appropriate concentrations of peptides in DMEM
with 0.5% FBS for the time indicated in the figures. A GFP tag was cloned 5’ into the
pEF5 vector in the KpnI and BamHI sites, generating the pEF5-GFP vector. cDNA
encoding for amino acids 163-468 of YopH was then cloned into the BamHI and EcoRI
sites of the pEF5-GFP vector. Jurkat transfections were performed as described(Vang,
Congia et al. 2005). Two days after transfection, dead cells were removed using the
Dead Cell Removal Kit from Miltenyi Biotec (Bergisch Gladbach, Germany).

4.3.8 Nucleofection
Jurkat cells were washed and resuspended in RPMI without phenol red to a
concentration of 50 x 10
6
cells/mL. A total of 5 x 10
6
cells/ sample were nucleofected in
the presence of the indicated peptides with an Amaxa Nucleofector® Device (Amaxa,
Walkersville, MD) using program X-005.

4.3.9 Flow Cytometry
Cells were diluted in HBSS without Ca
2+
and Mg
2+
, and supplemented with 5 mM EDTA
and 2.5 mg/mL BSA, and then analyzed using a BD FACSAria, BD LSR II, or BD
FACSCanto II (BD Biosciences, San Jose, CA). Data analysis and graph preparation
93
were conducted using FlowJo software (TreeStar Inc., Ashland, OR). GFP-positive cells
were gated by comparing the fluorescence of GFP-YopH transfected cells with the
fluorescence of untransfected cells, and using the gate subtraction feature of FlowJo.

4.3.10 Confocal Microscopy
After treatment with cell-permeable peptides, cells were washed and were viewed under
a Zeiss LSM 510 confocal microscope. COS-7 cells were plated in Lab-Tek II chamber
slides (Thermo Fisher Scientific, Rochester, NY) and allowed to adhere overnight in
culture medium. Jurkat cells were viewed after spontaneous or cyto-spin induced
adherence to a microscope slide.

4.3.11 High Content Imaging
RAW 264.7 cells were plated in 96-well BD imaging plates (50 x 10
3
cells/well) and
allowed to adhere overnight in culture medium. After incubation with the appropriate
concentrations of peptide, cells were washed and fixed for 15 min at room temperature
in 3.7% formaldehyde. After washing, cells were then incubated with Sytox Orange
(Invitrogen) and Cellmask Deep Red cytoplasmic/nuclear stain (Invitrogen). Automated
image acquisition was done using the Opera confocal reader (model 3842-Quadruple
Excitation High Sensitivity (QEHS), Perkin Elmer, Waltham, MA 02451). Images were
analyzed within the Opera environment using standard Acapella scripts. The algorithm
was used to identify objects such as nucleus based on Sytox Orange dye and cytoplasm
based on Cellmask Deep Red cytoplasmic/nuclear stain. Mean fluorescence intensity of
the cytoplasm was determined by analysis with Acapella software (Perkin Elmer). After
identification of nuclear regions by Sytox Orange staining, cytoplasmic regions were
calculated by subtracting the nucleus from the whole cell region.
94
4.3.12 Graphs and Statistics
Graphs, curve fittings and kinetic parameter calculations were performed using the
GraphPad Prism software package (GraphPad, San Diego, CA). All SD of differences
and ratios were calculated according to the error propagation rules described by
Taylor(Taylor 1997).

4.4 Results & Discussion
As a model system for our assays we used a 9 aa peptide derived from the sequence
surrounding the activating Tyr394 within the active site of Lck. Phosphorylation at this
site in T cells is physiologically controlled by PTPN22(Cloutier and Veillette 1999; Vang,
Congia et al. 2005) and SHP-1(Chiang and Sefton 2001), two PTPs which are highly
expressed in hematopoietic cells. Dephosphorylation of Lck Tyr394 also mediates the
pathogenic action of YopH, a PTP which is a virulence factor for the plague agent
Yersinia pestis(Alonso, Bottini et al. 2004). YopH and PTPN22 are considered drug
targets respectively for infectious diseases and autoimmunity(Vazquez, Tautz et al.
2007; Yu, Sun et al. 2007). Previously, we showed that the pCAP form of this peptide is
an excellent substrate of PTPN22(Mitra and Barrios 2007). The same peptide was also
dephosphorylated very efficiently by recombinant SHP-1 and YopH (k
cat
= 28.2 ± 2.2 and
K
M
= 67.2 ± 13.4 µM for SHP-1; k
cat
= 119.8.4 ± 2.9 and K
M
= 23.5 ± 1.6 µM for YopH,
(data not shown).

As a preliminary step to using pCAP peptides as intracellular probes for PTP activity, we
assessed whether CAP peptides can undergo in vitro phosphorylation by protein
tyrosine kinases (PTKs). We assessed the activity of recombinant Csk and Lck on a
variety of peptides including KKKKEEI-CAP-FFF (an optimized recognition sequence for
95
Csk(Sondhi, Xu et al. 1998)) and EEEI-CAP-GVLFKKK (an optimized recognition
sequence for Lck(Songyang, Carraway et al. 1995)). By monitoring fluorescence of the
peptide solution, we could not detect any activity of these PTKs on CAP peptides. We
could not detect significant decrease of 4-methylumbelliferone (MU) fluorescence as well
when incubated with the same PTKs (data not shown). Although we did not test for low
stoichiometry phosphorylation by alternate methods, we could conclude that the
phosphorylation of coumarin derivatives by PTKs is at the best an unfavorable reaction.
Next, we compared the ability of recombinant active serine phosphatases to
dephosphorylate DiFMUP and the 9LckpCAP394 (pCAP) peptide. Neither PP1, PP2A
nor PP2B showed any activity on the Lck pCAP peptide even at high enzyme
concentrations, despite using the optimal buffer for each enzyme. In the same buffer
each enzyme showed high activity on DiFMUP (Fig. 4.1B). We conclude that pCAP
peptides are not dephosphorylated by serine phosphatases in vitro.

Figure 4.1. pCAP peptides are specific PTP activity probes. (A) Structure of pCAP, CAP and Et 2pCAP.
(B) pCAP peptides are not dephosphorylated by serine-threonine phosphatases. Dephosphorylation of 0.2
mM pCAP peptide by PP1 (filled triangles), PP2A (open squares), or PP2B (filled squares). Linear
regression is shown. Open diamonds and dashed line show activity of enzymes on DiFMUP.

B A
96
In order to assess whether pCAP peptides could be used to detect intracellular PTP
activity, sea urchin oocytes, which express a whole range of PTPs, including homologs
to PTPN22 and SHP-1(Byrum, Walton et al. 2006) were microinjected with the pCAP
peptide. Dephosphorylation of the peptide resulted in a rapid increase in cell
fluorescence as visualized by microscopy (Fig. 4.2, top panel). To verify that
dephosphorylation of the peptide was the result of PTP activity, the fluorescence of cells
injected with the peptide was compared with that of cells which were co-injected with the
peptide and the specific PTP inhibitor sodium orthovanadate(Gordon 1991). Minimal
fluorescence in cells coinjected with the peptide and the PTP inhibitor vanadate
suggests that the pCAP peptides are dephosphorylated exclusively by intracellular PTPs
once they are delivered to the cell cytosol (Fig. 4.2, bottom panel).
Figure 4.2. Detection of intracellular PTP activity in microinjected cells. Images of sea urchin eggs
were taken 7’ after microinjection on a fluorescence microscope. Sea urchin cells were injected with pCAP
peptide dissolved in DMSO (upper panels) or in DMSO containing 10 mM vanadate (lower panels). To
correct for cell/injection volumes, and as an injection marker, fixed amounts of rhodamine were co-injected
with the peptides. Left panels shows fluorescence of the peptide as detected using a DAPI cube. Middle
panels shows fluorescence of the injection control as detected using a rhodamine cube. Right panels show
phase contrast microscopy. All images are in pseudocolor. Similar data were obtained by microinjecting
COS-7 cells (data not shown). Performed by Sagar Damle and Stephanie Stanford.

97
Next, we began optimizing the assay for use on mammalian cells, by delivering the
probes into whole populations of cells, while intracellular dephosphorylation of pCAP
peptides was quantitatively assessed by flow cytometry. Different concentrations of the
highly fluorescent 9LckCAP394 (CAP) peptide were delivered into Jurkat cells by
nucleofection. A dose-dependent increase in fluorescence proportional to the amount of
nucleofected peptide was observed (Fig. 4.3). The best signal to noise ratio was
obtained with a peptide concentration of 100 uM. These optimal nucleofection conditions
and peptide concentrations were then used to detect time-dependent intracellular
dephosphorylation of nucleofected pCAP peptides by flow cytometry. An increase in
fluorescence was observed within a minute after nucleofection of pCAP peptide in Jurkat
cells (Fig. 4.4A). No further increase in fluorescence was observed at longer incubation
time points. As controls, cells nucleofected with CAP peptide or with a non-hydrolyzable
version of pCAP in which the pCAP group was replaced by an Et
2
pCAP (see Fig. 4.1A),
showed only background fluorescence signal (Fig. 4.4A). To evaluate the contribution of
extracellular dephosphorylation of peptides adhering to the outer cell membrane surface,
we measured the fluorescence of cells incubated with the three peptides pCAP, CAP
and Et
2
pCAP without nucleofection. Cells incubated with CAP peptide showed increased
background fluorescence compared with cells incubated with either pCAP or Et
2
pCAP
but such fluorescence was consistently lower than the initial fluorescence of cells
nucleofected with pCAP, suggesting that non-specific binding of peptides to the cell
surface contributed minimally to cell fluorescence (Fig. 4.4A and Fig. 4.5). Importantly,
pre-treatment of cells with vanadate completely inhibited the increase in fluorescence of
cells nucleofected with pCAP, without affecting the fluorescence of cells nucleofected
with CAP peptide (Fig. 4.4B). A dose-response analysis showed that inhibition of
phosphatase activity could be detected by incubating cells with as low as 5 µM vanadate
98
(Fig. 4.4C). Overall, the data suggest that dephosphorylation of pCAP to CAP by
intracellular PTPs leads to a time-dependent increase in fluorescence of cells
nucleofected with pCAP peptides, which is detectable by microscopy and flow cytometry.

Figure 4.3. Optimization of CAP peptide nucleofection into Jurkat cells. Cells were nucleofected in the
presence of increasing amounts of CAP peptide and their fluorescence was measured by flow cytometry at
1’ after nucleofection. (A) Fluorescence of cells nucleofected with 100 µM CAP peptide (solid red line) or
DMSO (shaded gray graph). Dashed red line shows fluorescence of non-nucleofected cells incubated in the
presence of 100 µM CAP peptide (dashed red line). (B) Average fluorescence of non-nucleofected cells (red
circles, dotted line) or cells nucleofected with increasing amount of CAP peptide (red squares, continuous
line). Red lines are linear regressions. 95% confidence ranges for the measurements are shown in the graph
as black dotted (non-nucleofected cells) or continuous (nucleofected cells) lines.

A B
99
Figure 4.4. pCAP peptides can be used to monitor intracellular PTP activity. (A) Intracellular PTP
activity can be monitored by flow cytometry. Cells were nucleofected with 100 µM pCAP peptide (blue lines),
CAP peptide (red lines) or the non-hydrolyzable Et 2pCAP peptide (green lines). Dashed red line shows
fluorescence of non-nucleofected cells incubated with 100 µM CAP peptide. Graph shows fluorescence of
cells 30 sec (dotted lines) or 1 min (continuous lines) after nucleofection. (B,C) Inhibition of intracellular
pCAP dephosphorylation by the cell-permeable PTP inhibitor sodium orthovanadate. (B) Graphs show
fluorescence of cells 1 min after nucleofection with 100 µM pCAP peptide (blue lines) or CAP peptide (red
lines). Cells were preincubated for 60 min with 10 mM vanadate (dotted lines) or buffer alone (continuous
lines). Dashed lines show fluorescence of non-nucleofected cells co-incubated with 100 µM pCAP peptide
(blue) or CAP peptide (red) and 10 mM vanadate. Time course experiments also showed that the same
inhibition by vanadate could be achieved by a pre-treatment time of 5’ (data not shown). The fluorescence of
nucleofected cells incubated with vanadate is lower than fluorescence of non-nucleofected cells incubated
with vanadate, which is likely due to the lower size of nucleofected cells, thus leading to lower non-specific
binding of peptide to the extracellular surface. (C) Dose response of PTP inhibition by vanadate.
Fluorescence of cells 1 min after nucleofection with 100 µM pCAP peptide (blue squares). Cells were
preincubated for 5 min with increasing amounts of vanadate. Graph shows average +/- SD of nucleofected
cells (blue squares) and non-nucleofected cells (black square) incubated with 100 µM pCAP peptide.

A
B C
100
Figure 4.5. Fluorescence of non-nucleofected cells incubated in the presence of the CAP-based
peptides used in Fig. 4.4A. Graph shows fluorescence of non-nucleofected cells incubated with 100 µM
pCAP peptide (dashed blue line), 100 µM CAP peptide (dashed red line) or 100 µM Et 2pCAP peptide
(dashed green line).

To optimize spontaneous intracellular delivery in mammalian cells, the highly fluorescent
CAP peptide was conjugated at the C-terminus with the cell-permeable peptide (CPP)
tag ANT, with the peptide sequence RQIKIWFQNRRMKWKK derived from aa 43-58 of
the third helix of the Drosophila Antennapedia protein homeodomain(Sugita, Yoshikawa
et al. 2008). Incubation of Jurkat cells with this ANT-tagged CAP peptide did not result in
any detectable fluorescent cells, suggesting that the peptide tag was not efficient in the
intracellular delivery of the CAP peptide (data not shown). Prior studies have shown that
an 8-Arg peptide tag is very effective in driving cargo internalization into Jurkat T
cells(Mitchell, Kim et al. 2000). However, when Jurkat T cells were treated with CAP
conjugated with an 8-Arg peptide (R8) at its C terminus, internalization was slightly more
101
efficient, but still very weak. In a recent study, Pham et al. reported that N-terminal
conjugation of poly-Arg tags with saturated lipidic chains significantly increases their
carrier efficiency(Pham, Kircher et al. 2004). Thus we decided to test whether
conjugation of R7-CAP to lipidic chains with 14 or more carbon units would enable more
efficient internalization into cells. Fig. 4.6A shows that C14-R7-CAP was internalized in
Jurkat cells much more efficiently than R8-CAP. Lipidic chains longer than 14 carbons
did not increase efficiency but were rather associated with decreased internalization
efficiency (Fig. 4.6B). Confocal microscopy of Jurkat T cells incubated with C14-R7-CAP
showed that the peptide underwent intracellular internalization, and interestingly it
assumed a cytosolic localization (Fig. 4.6B, see also Fig. 4.8). Cytosolic localization of
the peptide is likely due to lipidic chain conjugation, which is known to affect the
subcellular localization of cell-permeable peptides(Pham, Kircher et al. 2004). We
reasoned that since Lck and the three above-mentioned PTPs targeting the Lck-Tyr394
residue are cytosolic proteins, cytosolic localization of the peptide might help reduce the
background dephosphorylation of the intracellular probe. The ability to control subcellular
localization of the activity probe can be particularly advantageous when the assay
focuses on sites that can be dephosphorylated by phosphatases residing in different
subcellular compartments (for example Tyr187 of the MAP kinase ERK2 is
dephosphorylated by HePTP in the cytosol and by VHR in the nucleus(Mustelin and
Tasken 2003)). CPPs can be internalized through endocytosis-dependent or
independent mechanisms(Joliot and Prochiantz 2004; Patel, Zaro et al. 2007). Co-
localization experiments using labeled dextran(Wittrup and Belting 2009) suggested that
C14-R7-CAP was internalized by endocytosis, followed by significant leakage into the
cytosolic compartment, where the probe becomes available for dephosphorylation by
102
PTPs (Fig. 4.7). For CPPs internalized by endocytosis, it has been shown that leakage
from endocytic vesicle is proportional to the peptide: lipid ratio(Fuchs and Raines 2004).
Figure 4.6. Optimization of cell-permeable pCAP peptides. (A,B) Flow cytometry analysis of uptake of
cell-permeable CAP by Jurkat cells. (A) Cells were incubated with 12.5 µM C14-R7-CAP (red line), CAP-R8
(yellow line), or with medium alone (gray shaded graph) for 1 h. Black dotted line shows fluorescence of
cells incubated with non-cell-permeable CAP (to correct for fluorescence due to non-specific binding to outer
membrane surface). (B) Cells were incubated with 10 µM C14-R7-CAP (red line), C16-R7-CAP (blue line),
C18-R7-CAP (green graph), C20-R7-CAP (pink graph) or with medium alone (gray shaded graph) for 1 h.
Black dotted line shows fluorescence of cells incubated with non-cell-permeable CAP. Inset shows confocal
microscopy of a representative Jurkat cell incubated with 10 µM C14-R7-CAP peptide for 1 h and visualized
using a DAPI cube.

Figure 4.7. Cytosolic distribution of C14-R7-CAP peptide. COS cells were incubated in medium
containing 12.5 µM C14-R7-CAP peptide and 2.5 mg/mL AF555-dextran as an endocytosis marker. Images
of cells were taken on a confocal fluorescence microscope after 1 h incubation. Figure shows CAP
fluorescence (left panel), dextran fluorescence (middle panel) and overlap (right panel). Notice the nuclear
exclusion and the cytosolic leakage (regions of no co-localization with dextran) of CAP fluorescence.

A B
103
Figure 4.8. Automated imaging of RAW cells. Cells were treated with 50 µM C14-R7-CAP peptide or
DMSO for 1 h and imaged using the Opera confocal reader. Fluorescence of CAP, nuclei, and whole cells
are shown in blue, green, and red, respectively.

104
To capture and analyze the internalization and subsequent intracellular
dephosphorylation of the peptide substrates by PTPs, a high-content image (HCI)-based
assay was developed (see Fig. 4.8 for a representative microscopic field). The
internalization event was monitored by incubating the mouse macrophage RAW 264.7
cells with different concentrations of the C14-R7 CAP peptide for time points ranging
from 0 to 60 minutes. Fig. 4.9A shows that internalization of the CAP peptide in RAW
cells varied in a non-linear manner with time of incubation and concentration of the
probe. Internalization of the probe was already evident at 5 min, however it tended to
plateau with time only at lower probe concentrations. Fig. 4.9B shows that in RAW cells
incubated with peptide probes for 1h, a dramatic increase in cytosolic fluorescence was
observed in cells incubated with concentrations of pCAP peptide above 50 µM. This
phenomenon was highly suggestive of intracellular dephosphorylation of the leaked
pCAP peptide, since it was not observed in cells incubated with the CAP version of the
probe, as an additional phenomenon was occurring with the pCP which was not
occurring with the CAP peptide. We concluded that cytosolic leakage of the peptide
followed by its intracellular dephosphorylation is dependent on the dose of the probe
according to non-linear kinetics. For our probe we determined that incubation of cells
with 100 µM or more probe for at least 30 min in Jurkat cells and 1 h in RAW cells
ensured a significant pCAP signal (data not shown). The signal was stable after cell
fixation (used for HCI). Probe concentrations and times of incubations needed in order to
detect dephosphorylation might need to be optimized in other systems depending on
probe sequence, cell type, and on the sensitivity of the approach used to detect cell
fluorescence (flow cytometry, microscopy or other).

105
B
Figure 4.9. Internalization of cell-permeable fluorogenic PTP activity probes. High content analysis of
dose and time-dependent internalization of C14-R7-CAP peptide. (A) Average cytoplasmic intensity of RAW
cells incubated with DMSO alone or increasing amounts of C14-R7-CAP peptide for 5’, 15’, 30’, and 60’.
Increasing fluorescence intensity was observed with increasing peptide concentration. Nonlinear fittings of
the data points are shown. (B) Intracellular dephosphorylation of cell-permeable pCAP peptide depends
upon peptide concentration. RAW cells were incubated for 1 h with 3 different concentrations of C14-R7-
CAP peptide (red line) or C14-R7-pCAP peptide (blue line). Graph shows relative cytoplasmic intensity of
cells where the intensity of cells treated with the lowest amount of peptide was used as a standard. Notice
the different increases in the slope of the connecting lines between the CAP and pCAP peptides when the
concentration of peptide increases.

106
To validate the optimized HCI assay for application in drug-discovery, RAW cells co-
incubated with the cell-permeable probe and vanadate showed inhibition of intracellular
dephosphorylation of the probe and the assay was sensitive to vanadate concentrations
as low as 100 µM (Fig 4.10A and 4.10B). Cellular toxicity as determined by reduction in
cell number was not observed with vanadate concentration as high as 1 mM. The
parallel analysis of cell samples incubated with control CAP peptide ensured that the
reduced fluorescence of cells incubated with vanadate was not due to effects of the
compound on peptide internalization. These studies suggest that pCAP-based peptides
could be used in cell-based high content imaging platforms to screen and identify PTP
inhibitors. Sensitive detection of inhibition of peptide dephosphorylation by vanadate was
similarly achieved in Jurkat cells co-incubated with pCAP peptide and various amounts
of vanadate (Fig. 4.11), suggesting that our assay is suited to flow-cytometry based
high-content platforms as well.

Two previously published cell-based assays detected the activity of a single PTP at the
cell-population level by subtracting the background activity of untransfected/uninfected
cells from the activity of cells overexpressing PTP-1B or CD45(Cromlish, Payette et al.
1999; Peters, Davis et al. 2003). To determine if our assay was suitable for detecting the
phosphatase activity of an overexpressed PTP at the single-cell level, Jurkat cells were
transfected with GFP-YopH and dephosphorylation of the pCAP peptide was measured
by flow cytometry. Preliminary analysis of these cells showed that while pCAP
fluorescence increased proportionally to GFP fluorescence in cells with low GFP
fluorescence, highly GFP-positive cells showed extremely low or no pCAP fluorescence
signal (data not shown). This could presumably be due to the toxic effects of GFP and/or
active YopH overexpression, thereby resulting in poor uptake of the peptide substrate.
107
A
B
However, if all the highly GFP fluorescent cells were gated out (Fig. 4.12A and 4.12B),
then increase in the fluorescence of the pCAP peptide was higher in the in GFP-positive
versus GFP-negative cells (Fig. 4.12C), strongly suggesting increased intracellular
dephosphorylation of the peptide in cells overexpressing YopH.
Figure 4.10. HCI assay of intracellular PTP activity using cell-permeable pCAP peptides. Detection of
PTP activity by high content analysis. (A) Average cytoplasmic intensity of RAW cells incubated for 1 h with
40 µM C14-R7-CAP peptide (red histograms) or 100 µM C14-R7-pCAP peptide (blue histograms) and with
increasing amounts of vanadate. (B) Microscopy of cells from two of the samples shown in panel (A).
Fluorescence of CAP and nuclei is shown in blue and green, respectively.

108
Figure 4.11. Flow cytometry assay of intracellular PTP activity using cell-permeable pCAP. Cells were
incubated for 1 h with 100 µM C14-R7-pCAP peptide (A) or C14-R7-CAP peptide (B) and with increasing
amounts of vanadate. Histograms show fluorescence of cells treated with peptide and with 25 µM (solid
black lines), 100 µM (dotted blue or red lines), 250 µM (dashed lines) and 500 µM vanadate (solid blue or
red lines), or with peptide alone (shaded graphs).

Figure 4.12. Single-cell assay of YopH activity. Jurkat cells were transfected with a plasmid encoding
GFP-YopH and incubated for 45 min with 100 µM C14-R7-pCAP peptide. (A) Dot plot of GFP fluorescence
v. FSC shows gating to exclude the high GFP+ cells, which were completely pCAP-negative (see text). (B)
Histogram of GFP fluorescence shows gating to separate GFP+ (green) and GFP- (orange) populations. (C)
Histogram shows CAP

A B
A
B
C
109
In conclusion, we show that pCAP-based peptides are excellent probes for monitoring
PTP activity at the single-cell level. The intracellular delivery of the pCAP peptide was
greatly improved by conjugation of C14-R7, a 14 carbon lipophilic chain attached to
seven arginine residues. The pCAP-peptide assay can be applied to live and fixed cells
and fluorescence can be detected by microscopy and quantitatively measured by flow
cytometry or HCI. To date, this is the first example of a cell-based assay to monitor
protein tyrosine phosphatase activity at the single-cell level. Single-cell imaging of PTP-
substrate intermediate formation has been recently reported by Yudushkin et al. using
an elegant FRET-based approach(Yudushkin, Schleifenbaum et al. 2007). Direct
detection of intracellular tyrosine kinase activity in single cells has been successfully
applied for high-throughput screening(Allen, DiPilato et al. 2006). pCAP peptides can be
developed into useful probes for secondary screening of candidate PTP inhibitors. With
the continuous increase in throughput of high-content approaches based on
microscopy(Bullen 2008), flow cytometry(Edwards, Young et al. 2009) and other
methods, the pCAP-based phosphatase assay offers significant advantages as a
primary screen to identify lead PTP inhibitors.

110
Chapter 5: Conclusions
5.1 LYP as a drug target for treatment of autoimmunity.
In the present work we have considered the lymphoid tyrosine phosphatase, a PTP with
a critical negative regulatory role on signaling through TCR. A single-nucleotide
polymorphism (SNP) C1858T in the PTPN22 gene recently emerged as a major
population-independent risk factor for multiple human autoimmune diseases(Bottini,
Vang et al. 2006). Recent genome wide association studies showed that in Caucasian
populations LYP ranks in second and third place in terms of single-gene contribution to
the genetic risk of RA and T1D, respectively, confirming its role as a major human
autoimmunity gene and its place as a drug target for prevention and early therapy of RA,
T1D and other autoimmune diseases(Todd, Walker et al. 2007).

Given the increased activity of the autoimmune-associated LYP-W620, we and others
have hypothesized that a selective small-molecule inhibitor of PTPN22 could be used as
a treatment for autoimmunity by reverting the gain-of-function effect of LYP-W620 and
restoring normal levels of TCR signaling in autoimmune patients carrying the W620
variation(Vang, Congia et al. 2005; Rieck, Arechiga et al. 2007; Aarnisalo, Treszl et al.
2008). However, until now the molecular mechanism by which the W620 variant leads to
gain-of-function was unknown. Here we provide the first report of the molecular
mechanism by which the polymorphism leads to increased enzymatic activity of the
phosphatase and subsequently reduced TCR signaling. Our findings confirm the
previous report that the W620 variation leads to increased enzymatic activity of the
phosphatase(Vang, Congia et al. 2005), and support the notion that LYP could be a
druggable phosphatase, as the gain-of-function phenotype could be directly modulated
by a chemical inhibitor. Additionally, both the benign phenotype of the Ptpn22 -/-
111
mouse(Hasegawa, Martin et al. 2004), as well as the autoimmune-protective effect of the
loss-of-function R263Q variant(Orru, Tsai et al. 2009) also suggest that pharmacological
inhibition of LYP is unlikely to cause serious side-effects and may indeed provide
therapy for autoimmunity.

In addition to the genetic evidence of the role of LYP in autoimmunity, there is also
increasing awareness that reduced TCR signaling plays a pathogenic role in
autoimmunity(Sakaguchi, Takahashi et al. 2003; Siggs, Miosge et al. 2007). The TCR-
stimulating monoclonal antibodies against CD3 have been successful in trials of T1D,
further supporting the concept that positive modulation of the TCR helps treat
autoimmunity in at least a subset of patients(Chatenoud 2006). This suggests that
chemical inhibition of LYP, which would lead to increased TCR signaling, might also be
effective at treating autoimmunity in a variety of patients, and not solely those carrying
the W620 polymorphism.

5.2 The regulation of LYP in T cells
Here we report that the increased activity of LYP-W620 is due to reduced
phosphorylation on an inhibitory tyrosine residue, Y536. We report that this
phosphorylation is predominantly mediated by the Src-family kinase Lck, which is
recruited to LYP by the interaction of LYP with Csk. We have also shown that the kinase
activity of Csk is necessary for this recruitment, suggesting that phosphorylation of LYP
on additional site(s) by Csk may mediate the interaction between LYP and Lck. The
W620 variation disrupts the interaction between LYP and Csk, leading to reduced
recruitment of Lck to LYP, and reduced phosphorylation on the inhibitory site.

112
Exploration of additional post-translational modifications of LYP will provide further clarity
regarding the regulation of LYP, as well understanding the role of the interaction of LYP
with both Csk and Lck. Given that mutation of LYP at Y536 does not completely abolish
phosphorylation, nor the difference in phosphorylation between R620 and W620, we
believe there are additional sites which may be important for regulation of the the
phosphatase. In addition, the polymorphism could affect the interaction of LYP with other
proteins, its recruitment to specific subcellular fractions and/or its protein stability.

5.3 New assays for PTPs
While our focus has been to develop assays for PTPs which are drug targets for
autoimmunity – for example LYP and CD45 – other PTPs such as LMPTP, PTP1B,
SHP2, PRL3 and STEP have emerged as important drug targets for cancer, metabolic
diseases and Alzheimer’s(Iversen, Andersen et al. 2000; Bottini, Bottini et al. 2002;
Kikawa, Vidale et al. 2002; Ivins Zito, Kontaridis et al. 2004; Snyder, Nong et al. 2005;
Liang, Liang et al. 2007).
,
However, development of specific, cell-permeable PTP
inhibitors is currently hampered by the inappropriateness of current methods for high-
throughput screening (HTS). Although inhibitors of PTPs have been heavily sought after,
traditional enzymatic screening approaches have failed to produce anti-PTP drugs. The
main challenges have been the lack of cell-permeability and specificity of lead
compound inhibitors. The active site of PTPs is highly charged and shows a high degree
of identity between multiple members of the family. Potent inhibitors identified in
enzymatic HTS tend to be charged molecules with a low cell-permeability and low
specificity profile. The substrates used in these assays interact almost exclusively with
the active site of the enzyme, and traditional HTS approaches using these substrates
tends to identify active site inhibitors, which translates into a low specificity and high
113
charge of lead compounds. Our objective was to develop novel assays which can be
used to identify cell-permeable inhibitors of PTPs.

Our first approach was to use a novel enzymatic screen to identify non-phospho-mimetic
inhibitors of LYP. By choosing a library enriched with compounds with “drug-like”
properties, and by selecting screening conditions to minimize hits from competitive
inhibitors, we were able to identify several non-phospho-mimetic LYP inhibitors, one of
which was cell-permeable, able to increase T cell signaling and T cell activation, and
displayed characteristics of non-competitive inhibition. Given the high charge of the
active site of PTPs, using a screening approach which avoids the identification of
competitive inhibitors maximizes the chance of avoiding compounds which bind to the
highly charged active site, and thus increases the potential of finding uncharged
compounds which are cell-permeable. Non-competitive inhibitors also have the
advantage that their potency does not generally decrease in the presence of high
concentrations of substrate(Copeland 2005). This can be advantageous when using
compounds in vivo, as competitive inhibitors can lose potency if the inhibitor causes an
increase in the local concentration of substrate(Copeland 2005). Additionally, as the
active site of PTPs is highly conserved, allosteric inhibitors may also offer the advantage
of increased enzyme selectivity.

Our second approach was to develop a cell-based assay which can be used to screen
for cell-permeable PTP inhibitors. Here, we report the first cell-based tyrosine
phosphatase activity assay which can be used to directly monitor the activity of a
particular PTP at the single-cell level. Currently there is no high-throughput cell-based
assay to directly monitor the activity of PTPs. Our assay can be easily applied to any
114
PTP of interest through cell overexpression systems and optimization of the peptide
substrate sequence. When used as a first-pass screening, such an assay has potential
to identify new classes of lead compounds against PTPs, which are already optimized
for cell permeability and minimal cellular toxicity. Here we provide novel approaches to
identify inhibitors of LYP which can be extended to many PTPs of pharmaceutical
interest.
115
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Abstract (if available)

Abstract PTPs involved in modulation of signal transduction through the T cell receptor (TCR) are promising targets for human autoimmunity. Here we will focus on the lymphoid tyrosine phosphatase LYP, a critical negative modulator of TCR signaling encoded by the PTPN22 gene. A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for multiple human autoimmune diseases. In T cells, LYP forms a complex with the negative regulatory kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T SNP results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits reduced interaction with Csk and is a gain-of-function inhibitor of TCR signaling. While strong genetic and functional evidence has suggested that LYP is a promising candidate drug target for treatment of human autoimmunity, the molecular mechanism of the autoimmune-associated R620W variation remains unknown.

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Creator Stanford, Stephanie Michele (author)

Core Title The molecular mechanism underlying the autoimmune-associated PTPN22 R620W variation and the quest for therapeutics

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 08/10/2010

Defense Date 04/26/2010

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

Tag autoimmunity,cell-based assay,Csk,lymphoid phosphatase,OAI-PMH Harvest,PTP,PTPN22,TCR signaling,tyrosine phosphatase

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Bottini, Nunzio (committee chair), Allayee, Hooman (committee member), Hacia, Joseph G. (committee member), Stallcup, Michael R. (committee member)

Creator Email sbond@usc.edu,stephanie@liai.org

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3388

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