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Regulation of Aurora kinase B and its effect on phosphorylation of G9a/GLP
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Regulation of Aurora kinase B and its effect on phosphorylation of G9a/GLP
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Content
1
Master’s Thesis on
REGULATION OF AURORA KINASE B
AND ITS EFFECT ON
PHOSPHORYLATION OF G9a/GLP
by
Sushanth Adusumilli
Biochemistry and Molecular Biology
Master of Science (MS)
University of Southern California
August 2018
2
CONTENTS
• INTRODUCTION . .………………………………………………………………………………......3
o GC Signaling ....................................................................................3
a. Hormones and their Receptors ……………………………...................3
b. Glucocorticoids (GC) ..................................................................3
c. Mechanism of GC mediated Cell Signaling ................................4
d. Coregulators G9a & GLP ............................................................6
e. Mechanism of G9a/GLP coactivator function ...........................7
o Aurora Kinase B ..............................................................................8
a. Aurora Kinase B Structure .........................................................8
b. Aurora Kinase B Regulation .......................................................9
o Project Objectives .........................................................................11
• RESULTS .................................................................................................12
• DISCUSSION ...........................................................................................22
• MATERIALS AND METHODS ....................................................................25
• REFERENCES ...........................................................................................29
3
INTRODUCTION
I. GC SIGNALING
a. Hormones and their Receptors
Hormones play major roles in the transcriptional regulation of many genes. They are a
class of signaling molecules that are secreted by endocrine glands and transported by the
circulatory system to distant target organs to regulate their physiology and behavior. Hormones
have very diverse chemical structures and are classified into 3 major types: Steroids, eicosanoids
and Amino acid/Protein derivatives (small peptide hormones). Eicosanoids are derived from
lipids such as Arachidonic Acid and Prostaglandins. Steroid hormones are derived from
Cholesterol. Most hormones regulate the behavior of the organs/tissues by binding to their
specific receptors and then initiate different signal transduction cascades inside the cells. A single
hormone can initiate different pathways depending on the physiological conditions. Many
studies have showed cross-talks occurring between different hormone mediated cell signaling
pathways.
Hormones initiate cell signaling pathways through their specific receptors. These
receptors are generally classified into two types: cell-membrane associated and intracellular
receptors. The chemical nature of hormones, dictate the method of transportation and the type
of receptor at the target organ. As they are derived from Cholesterol, steroid hormones are
hydrophobic in nature and require carrier proteins for their transportation to target site. They
can easily diffuse through the lipid bilayer into the cytoplasm and bind to intracellular steroid
receptors to initiate cellular responses.
Hormones regulate a wide array of processes in the body like metabolism, reproductive
cycle, immune system, sensory perception, tissue function, respiration. They control the internal
environment of the body through homeostasis. The rate of hormone biosynthesis and secretion
is regulated by a homeostatic negative feedback control mechanism. The factors of such
mechanism would be plasma concentrations of ions or nutrients, environmental changes, mental
activity. The negative feedback cannot be triggered by the higher concentrations of hormone
alone but by overproduction of the “effect” of the hormone.
b. Glucocorticoids (GC)
My work is focused on a type of hormones called Glucocorticoids, a sub-class of steroids.
The word Glucocorticoids is a combination of Glucose and Cortico-Steroids. These steroid
hormones play a significant role in the metabolism of Glucose and they are secreted by the
Adrenal cortex gland. Cortisol is the most important human Glucocorticoid. The secretion of
these hormones is controlled by Hypothalamic – Pituitary – Adrenal (HPA) axis
[1]
. Certain signals
4
trigger the hypothalamus to release corticotropin releasing hormone (CRH), which acts on the
pituitary to stimulate the secretion of Adrenocorticotropic hormone (ACTH). ACTH then acts on
the adrenal cortex to stimulate the production and secretion of glucocorticoids. Glucocorticoid
functions can be classified into two major categories: Immunologic and Metabolic. The
immunologic effects would be down-regulation of pro- and up-regulation of anti-inflammatory
proteins respectively. They are known to play a role in brain development and homeostasis of T-
lymphocytes
[13][14]
. The metabolic effects would be mobilization of amino acids, stimulation of
gluconeogenesis, fat breakdown in adipose tissue, inhibition of glucose uptake in muscle and
adipose.
Due to their diverse influence from metabolism through cardiovascular system to bone-
metabolism and even central nervous system, they are widely used as therapeutic agents for
many medical conditions. They are primarily used to treat allergies, inflammatory and auto-
immune disorders, asthma and pain relief in inflammatory conditions
[15][16]
. Glucocorticoids
cause immunosuppression by decreasing the functions and numbers of lymphocytes.
c. Mechanism of GC mediated Cell-Signaling
Glucocorticoids, because of their steroidal structures, can diffuse through the lipid bilayer
into the cytosol. In the cytosol, they act as ligands to Glucocorticoid Receptor (GR). Originally, GR
is present in the inactive conformation in the cytosol and complexed with chaperone proteins
Hsp90, Hsp70 and c-Src protein
[1]
. Binding of GC ligand to GR triggers the conformational change
of GR into active form and dissociation of the complex. Then GR dimerizes and gets translocated
into the nucleus through the nuclear pore complex (NPC). Inside the nucleus, GR can bind directly
to the Glucocorticoid Response elements (GRE) on the genome and recruit a set of coregulator
molecules to promote or inhibit the assembly of DNA Polymerase II complex at the promoter
region and thus regulate transcription (Figure 1a). The same coregulator that acts as coactivator
to one gene can act as corepressor to another gene thus denoting their gene-specific action.
Once inside nucleus, GR can also regulate the transcription of target genes by physically
interacting with other transcription factors. The association of GR with specific members of the
STAT family has been shown to enhance the transcription of responsive genes. In contrast, the
interaction of GR with the transcription factors, AP1 and NF-κB
[19]
, antagonizes their activity. For
some genes, the response is accomplished by GR tethering to these DNA-bound proteins without
directly interacting with the DNA (Figure 1b). For other genes, however, GR functions in a
composite manner, binding directly to a GRE and physically associating with AP1 or NF-κB bound
to a neighboring site on the DNA (Figure 1c). As usually, they interact with coregulators to
regulate transcription.
5
Figure 1
[1]
. Classical Glucocorticoid Receptor (GR) signaling pathways – GR initially exists in
inactive form and complexed with chaperone proteins Hsp90 and Hsp70 and Src protein. Glucocorticoids
(GC) diffuse through the cell membrane into cytoplasm and bind to GR. GC ligand then triggers
conformational change in GR and the GR dissociates from chaperones and Src proteins making it active.
The active GR undergoes dimerization and gets translocated into the nucleus through Nuclear Pore
Complex (NPC). Inside the nucleus, GR can act in three different pathways: a) Direct – GR dimer can directly
bind to Glucocorticoid Response elements (GRE) in the genome and recruit coregulators to either promote
or suppress the assembly of RNA Polymerase II complex (Pol II) thus either causing activation or repression
of target genes. b) Tethering – Instead of directly binding to DNA, GR dimer attaches or ‘tethers’ to other
transcription factors like STAT dimer and NF-Κb and regulate the transcription of target genes by recruiting
coregulators and controlling assembly of Pol II complex. c) Composite – In addition to binding directly to
GRE, GR also physically interacts with other transcription factors like STAT dimer and AP1 to regulate the
transcription of target genes
Other than the above discussed ‘classical’ signaling pathways, evidence suggests that GR can act
via non-genomic mechanisms to regulate responsive genes. The c-Src protein once released from
GR complex, can activate different Kinase cascades like MAPK, PI3K and AKT that lead to the
6
phosphorylation of annexin 1, inhibition of cytosolic phospholipase A2 activity, and impaired
release of arachidonic acid leading to cellular responses
[17][18]
. The existence of non-genomic
signaling adds greater complexity and diversity to glucocorticoids and their biological actions
d. Coregulators G9a & GLP
Among numerous coregulators of GR, we are working on Euchromatin Histone Lysine
Methyltransferase 2 (EHMT2), also known by the name G9a. As the name suggests, it primarily
catalyzes the di-methylation of H3K9 (Lysine 9 on Histone 3) in Euchromatin
[3][20]
. S-Adenosyl
Methionine (SAM) serves as a cofactor and methyl group donor for histone methyltransferases
[20][21]
. Dimethylation at K9 on Histone 3 tail is required before trimethylation of H3K9 which is a
classical signal for transcriptional repression across the genome. The methylation causes the
compaction of chromatin by recruiting specific proteins like Heterochromatin Protein 1 complex
(HP1) and makes the DNA inaccessible for transcription. Apart from Histones, G9a is reported to
methylate other substrates
[2][22]
.
G9a has been observed to be upregulated in different types of cancer and its
overexpression has been associated with poor prognosis
[3]
. Key roles played by these enzymes
in various diseases have led to the hypothesis that these molecules represent valuable targets
for future therapies. Several small molecule inhibitors have been developed to specifically block
the epigenetic activity of these enzymes, representing promising therapeutic tools. Elevated G9a
levels were commonly correlated with higher methylation levels, leading to the suppression of
important tumor suppressor genes
[3]
.
Generally, lysine specific-Histone methyl transferases are subdivided into two classes: SET
domain and non-SET domain
[21]
. G9a falls under the SET domain class.
Figure 2. Domain Structure of G9a (isoform a)
[5]
– The N-terminal Transactivation Domain (TAD)
facilitates interaction with proteins like transcriptional coregulators and plays a significant role in the
coregulator function of G9a. The C-terminal Pre-SET, SET and Post-SET domains together make up the
catalytic core of G9a and catalyzes mono- and di-methylation of Lysine 9 on Histone 3 (H3K9). The Ankyrin
repeats (ANK) are special protein – protein interaction motifs which are common in many proteins. They
also read the methylation of H3K9 substrate for G9a.
7
The SET domain (Su(var)3-9, Enhancer of Zeste, Trithorax), contains the core catalytic
center of the lysine methyltransferases. It is flanked by pre-SET and post-SET domains on either
side and together they make up the catalytic domain of the protein. The pre-SET region contains
cysteine residues that stabilizes the Zinc fingers in the Proteins
[20]
. The SET domain is rich in β-
sheets. Often, the β-strands found in the pre-SET domain will form β-sheets with the β-strands
of the SET domain, leading to slight variations to the SET domain structure which alter the target
residue site specificity for methylation. This interplay between the pre-SET domain and the
catalytic core is critical for enzyme function. For G9a, the catalytic domain is present at the
carboxy terminal of the protein.
A G9a like Protein (GLP) has been identified, which has similar structure, properties and
function of G9a. GLP has unique substrates in addition to a set of common substrates with G9a.
G9a and GLP can form a homomeric and heteromeric complex via their SET domains. However,
endogenous proteins exist exclusively as the stoichiometric G9a–GLP heteromeric complex in
various human and mouse cells
[23]
. G9a forms a stoichiometric complex not only with GLP but
also with Wiz, a protein with multiple zinc finger motifs. Knockdowns of either GLP or Wiz is
naturally accompanied by reduction in G9a protein levels
[7]
which shows the implication of
heteromer complex formation in the stability of G9a.
e. Mechanism of G9a/GLP Coactivator function
The N-terminal Transactivating Domain (TAD) of G9a plays a significant role in its
coregulator function by interacting with other transcriptional coregulators. Lysine 185 in the TAD
of the human isoform a gets automethylated
[2][4]
. Methylation of K185 residue creates a binding
site for Heterochromatin Protein 1 gamma (HP1γ) protein specifically. Phosphorylation of the
adjacent Threonine 186 residue is catalyzed by Aurora Kinase B and it eliminates binding site for
HP1γ on G9a
[2][4]
. Similarly, GLP undergoes post translational modifications at K205
(automethylation) and T206 (phosphorylation) residues and shares similar binding properties
with HP1γ as G9a. The sites of methylation and phosphorylation on G9a/GLP has the sequence
A-R-K-T which is very similar as the Histone 3 (H3) site A-R-K (9)-S (10), another substrate of
G9a/GLP and Aurora Kinase B. H3K9 methylation by G9a/GLP creates binding site for HP1
proteins, whereas H3S10 phosphorylation by Aurora Kinase B abolishes binding site for HP1
proteins on Histone H3 which is similar to G9a-HP1γ interactions. Although, not involved in the
transcriptional repression of genes via Chromatin remodeling, these post translational
modifications on G9a/GLP are crucial for their coactivator functions as elaborated below.
Once activated by Glucocorticoids, GR binds to GR Binding regions (GBR) on DNA and
recruits G9a and GLP in A549 cells. G9a facilitates recruitment of GRIP1, p300 and Carm1
coactivators
[6]
, which acetylate Histones H3 and H4 and demethylates H3 at R17 respectively. If
G9a and GLP are auto-methylated, then it interacts with HP1γ protein (also known by the name
CBX 3, Chromobox Protein homolog 3)
[9]
. HP1γ which is phosphorylated on Serine 93 (pS93) then
interacts with the C-terminal tail of RNA Polymerase II (Pol II) with phosphorylated Serine 5 (pS5)
and promotes Pol II recruitment more efficiently to Promoter region of G9a/GLP dependent GR
target genes (Figure 3). It is interesting to note that pS5 on C-terminal of Pol II is widely
8
considered as a classical sign for Transcriptional Initiation. However, when G9a/GLP is
phosphorylated by Aurora Kinase B, HP1γ recruitment by G9a/GLP is blocked
[9]
and thus Pol II
cannot efficiently bind to promoter regions of the G9a/GLP dependent GR regulated genes
(Figure 3).
Figure 3. Mechanism of G9a/GLP coactivator function
[9]
– Once Glucocorticoid Receptor (GR)
dimer binds to distal GR Binding Regions (GBR), it recruits G9a/GLP heterodimer. G9a forms a coregulator
complex with other transcriptional regulators like GRIP1, Carm1 and p300. Usually, Carm1 demethylates
Histones and p300 acetylates Histones which act synergistically with their coactivator function. If G9a and
GLP are automethylated, then Heterochromatin Protein 1 gamma (HP1γ) binds to the methylated site. As
HP1γ binds to G9a/GLP, its orientation facilitates interaction of phosphorylated Serine 93 (not labeled) on
HP1γ and phosphorylated Serine 5 (not labeled) on RNA Polymerase II (RNA Pol II) promoting recruitment
of RNA pol II complex to the promoter region of G9a/GLP dependent GR target genes. This causes
upregulation of target genes like E-cadherin, which causes biological impact like changes in cell migration
properties. When G9a/GLP is phosphorylated by Aurora Kinase B, it abolishes the binding site for HP1γ and
thus blocks the recruitment of RNA Pol II complex.
II. AURORA KINASE B
a. Aurora Kinase B Structure
Aurora kinase B that catalyzes G9a/GLP phosphorylation is a member of family of novel Ser/Thr
Kinases, with Aurora Kinase A and C, which are crucial in many events across Mitosis and cell-
cycle control. They are found to be conserved during eukaryotic evolution, overexpressed in
tumor cells and might be involved in tumorigenesis
[12]
.
9
Figure 4. Domain structure of Aurora Kinase B
[12]
– The blue colored N-terminal domain is the
regulatory domain while the large green colored and C-terminal small blue colored domains make up the
Catalytic domain. The catalytic domain consists of an Activation Loop, where Threonine 232 (T232) plays
a significant role in the activation of catalytic domain as the name suggests. The destruction box or D-box
and A-Box are special motifs, that are involved in targeted degradation of Aurora Kinase B via Ubiquitin –
Proteasome pathway.
The Aurora Kinases are made up of two domains, a regulatory N terminal domain which
is diverse among the members and a catalytic C-terminal domain which shares almost 70%
homology among different members
[12]
.
Even though they have similar structures and domains, Aurora Kinases differ widely in
terms of substrates, functions and localization. Expression of Aurora Kinase B is induced in the
late G2 phase. Activated Aurora-B has a general chromosomal localization in G2 and prophase,
moves to chromosomal centromeres during prometaphase and metaphase, and subsequently
relocates to the spindle midzone during anaphase and telophase. Aurora-B forms a tight complex
with INCENP, Survivin and Borealin proteins within the cell during mitosis and are named as
chromosomal passengers
[12]
. Aurora Kinase B is known to be involved in Phosphorylation of
Histone proteins as previously mentioned. Also, Aurora Kinase B is found to play an essential role
in Chromatid separation and Cytokinesis. Inactivated Aurora-B is frequently associated with
multinuclearity in cells as its crucial in the cleavage furrow formation in Cytokinesis.
b. Aurora Kinase B Regulation
Aurora Kinases are regulated by two major mechanisms: Degradation and
phosphorylation. The D-Box and A-Box mentioned previously help in the targeted degradation of
the proteins by the Ubiquitin – Proteasome system, specifically via a ubiquitin-protein ligase,
Anaphase promoting complex or cyclosome (APC/C)
[28][29]
. Aurora Kinases undergo
autophosphorylation by interacting with other passenger proteins which help in induction of
active confirmation of the catalytic domain.
Aurora Kinase B is known to associate with INCENP (inner centromere Protein). This
association stimulates the Kinase activity of Aurora Kinase B and causes conformational changes
in the N-terminal and C terminal domains, which leads to autophosphorylation of T-232
[11]
. The
phosphorylation of T-232 is required for the activity of Aurora Kinase B and is reported to be
highly correlated to mitosis, specifically the phosphorylation of its substrates Histone H3 and
vimentin
[11]
. It is also reported that the phosphorylation of IN Box in C-terminal of INCENP is
involved in the modulation of Aurora Kinase B activity and phosphorylation of T-232
[11]
. The IN
10
Box domain contains the binding domain for Aurora Kinase B and gets phosphorylated by Aurora
Kinase B, once its kinase activity gets stimulated by binding to INCENP. It has been reported that
there is an effect on their binding affinities due to phosphorylation of Aurora Kinase B and
INCENP.
Other studies have showed that Aurora Kinase B gets phosphorylated at Serine 331 by
Checkpoint Kinase 1 (Chk1) in prometaphase, a prerequisite for optimal phosphorylation of TSS
Motif containing IN Box of INCENP, that are essential for the complete activation of Aurora Kinase
B
[10]
. It is reported to be the first non-T loop phosphorylation found and that it has no effect on
binding of INCENP to Aurora-B and autophosphorylation of T-232. Also, phosphorylation of
Ser331 is required for Survivin association with the chromosomal passenger complex and
localization of Aurora-B to the kinetochores
[10]
.
Thus, the proposed model of activation of Aurora-B is that Aurora-B associates with
INCENP and Survivin (not shown in Figure 5) in the complex first and T-232 gets auto
phosphorylated leading to partial activation. Then during prometaphase, Chk1 phosphorylates at
Ser331 which leads to phosphorylation of TSS motif on INCENP which then leads to complete
kinase activation
[10]
. In contrast, it has been reported that Ser331 is phosphorylated via an
unknown kinase during prophase, anaphase and cytokinesis independently of Chk1.
Figure 5. Mechanism of Aurora Kinase B (AurKB) activation – Aurora Kinase B, by itself exists in
inactive form. AurKB associates with Inner Centromere Protein (INCENP), which causes conformational
changes and lead to autophosphorylation of Threonine 232 (T232p), making it partially active. Then, AurKB
is phosphorylated by Checkpoint Kinase 1 at Serine 331 (S331p), which causes it to phosphorylate TSS motif
(Threonine – Serine – Serine) on INCENP, marking the complete activation of AurKB.
Recent studies showed that Aurora-B activity does not significantly change throughout
mitosis
[24]
and is backed up by the fact that Ser331 is constitutively phosphorylated across
different conditions
[10]
. It is known that Protein phosphatases – Protein Phosphatase 1 (PP1) and
Protein Phosphatase 2 (PP2) oppose Aurora-B mediated signaling
[25][26]
. Recently, Sds22, a known
regulator of PP1 is shown to regulate Aurora-B activity
[27]
.
Other studies have shown that End Binding Protein 1 (EB1) promotes Aurora Kinase B
activity by blocking its inactivation by PP2
[8]
. EB1 plays an important role in microtubule dynamics
11
and colocalizes with Aurora B on the central spindle in anaphase and at the midbody in
cytokinesis
[8]
. EB1, although not a substrate, interacts with Aurora-B and the proposed model is
that it protects T-232 from Dephosphorylation, specifically by PP2 but not by PP1 (Figure 6). This
denotes the specificity and complexity in the Aurora Kinase mediated signaling. EB1 does not act
as a direct inhibitor of PP2 as it cannot protect Aurora-A from dephosphorylation by PP2
[8]
.
Figure 6. Mechanism of Aurora Kinase B (AurKB) inactivation – Aurora Kinase B is inactivated by
the action of Protein Phosphatase 1 (PP1) and Protein Phosphatase 2 (PP2). Both PP1 and PP2 can
dephosphorylate Threonine 232 (T232p) and Serine 331 phosphates (S331p) as they are general
phosphatases. End Binding Protein 1 (Eb1) plays a unique role in this mechanism where it shields only
T232p from dephosphorylation specifically by PP2. Eb1 has no effect on dephosphorylation of both T232p
and S331p by PP1 and S331p by PP2.
PROJECT OBJECTIVES
Precursor B-cell acute lymphoblastic leukemia (B-ALL) is a form of blood cancer in which
too many B-cell lymphoblasts (immature B cells) are found in blood and bone marrow.
Glucocorticoids are widely used as essential components in the therapy of B-ALL for a long time
[32]
. Results from Dr. Stallcup’s lab indicate that G9a/GLP coactivator function is implicated in
activation of genes causing GC induced cell death in B-ALL cells. In addition, Aurora Kinase B is
significantly overexpressed in relapsed B-ALL cells in comparison with diagnosis and treatment
with Aurora Kinase B inhibitor sensitize the B-ALL cells to GC induced cell death. Aurora Kinase B
activity is regulated via different mechanisms. Thus, the goal of my project is to study the effect
of these specific Aurora Kinase B regulators: Chk1, EB1, PP1 and PP2 on the phosphorylation of
12
G9a/GLP complex and its coactivator function. By understanding Aurora Kinase B regulation and
its effect on phosphorylation of G9a/GLP, we hope to use the knowledge gained, in designing
improved therapies for B-ALL.
RESULTS
Silencing of PP2a reduces phosphorylation of overexpressed G9a in Cos7.
Since Chk1, PP1a, PP2A and Eb1 are known to regulate Aurora Kinase B activity, we
hypothesized that changes in their expression would causes changes in the amount of G9a
phosphorylation by modulating Aurora Kinase B activity. So, to study the effect of the Aurora
Kinase B regulators on G9a phosphorylation, our approach was to co-transfect validated siRNA
(Figure. 1) for each regulator and HA tagged G9a overexpression plasmids. Cos7 cell line was
chosen for these experiments as the overexpression and knockdown of proteins are easier to
perform and more efficient. Whole cell extracts were then immunoprecipitated with pan
phospho-threonine antibody. The reason behind this step is that phospho-threonine antibody
captures the changes in the phosphorylation of overexpressed G9a when regulators are knocked
down compared to controls. It can then be visualized by immunoblotting with HA antibody.
Figure 1: Validation of siRNA.
Cos7 cells were transfected with siRNA for the regulators of Aurora Kinase B separately (siChk1,
siEb1, siPP2a, siPP1a). Lysates were then immunoblotted with the antibody for the protein targeted by
the transfected siRNA along with loading control Actin.
We show that when PP2a is silenced, the amount of overexpressed G9a
immunoprecipitated with phospho-threonine antibody is significantly reduced when compared
to control siNS (Figure 2a, top panel, third lane). This means that silencing of PP2a reduces
phosphorylation of overexpressed G9a. This was replicated over a total of two independent
experiments. This is an unexpected result, since PP2A is known to deactivate Aurora Kinase B and
thus when knocked down, we would expect Aurora Kinase B to be more active and cause more
13
G9a phosphorylation. In addition, when PP1a and Eb1 were silenced, no significant changes were
observed in the amount of phosphorylated G9a (Figure 2a, top panel second lane and 2b, top
panel third lane) compared to control SiNS and this held true for over two and three independent
experiments respectively.
Figure 2: siRNA knockdown of PP2a reduces phosphorylation of G9a in Cos7
Co-transfection of HA tagged human G9a Full Length Wild type plasmid (HA hG9a FL WT) and
siRNA for regulators of Aurora Kinase B ((a) – siPP1a, siPP2a; (b) – siChk1, siEb1) in Cos7 cells. Lysates were
then Immunoprecipitated with phospho-threonine Antibody (IP phospho Threonine) followed by
immunoblotting with HA Antibody. Immunoblots were also performed separately on Lysates for loading
and transfection controls. n – number of independent experiments.
Silencing of Chk1 causes downregulation of G9a in Cos7.
When Chk1 is silenced, unexpectedly we observed significant down-regulation of input
overexpressed G9a when compared to control SiNS over three independent experiments (Figure
2b, bottom panel, middle lane and Figure 3, Panels 2-4). Interestingly, we also observed down-
regulation of endogenous G9a when Chk1 is silenced in all the three independent experiments
(Figure 3). In figure 3, first panel, the gel was run for longer time than required to achieve a visible
separation between overexpressed G9a and endogenous G9a. Since overexpressed G9a has a
HA-tag, its band rests slightly higher than the endogenous G9a protein (Figure 3), which is also
accompanied by an immunoblot with HA antibody on the right side. Comparing the lanes with
Chk1 knockdown to the control (Figure 3, panel 1 - lanes 2,4,6), we conclude that Chk1 is
implicated in the stability of G9a protein and knocking it down is not a suitable strategy to
investigate the regulation of G9a phosphorylation.
14
Figure 3: siRNA knockdown of Chk1 reduces G9a stability
Co-transfection of HA-tagged G9a and siChk1 in Cos7 cells. Immunoblots were performed on
lysates for loading and transfection controls (panels 2-4), represented for all the independent experiments.
Lysates from three independent experiments were run on a gel for longer time than usual and then
immunoblotted with G9a and HA to separate overexpressed G9a (Ov) from endogenous G9a (En).
Inhibition of PP2A causes overexpression of G9a/GLP and AurKB proteins in Nalm6
After showing that silencing of PP2a leads to reduction in the Phosphorylation level of
overexpressed G9a in Cos7, we decided to focus on PP2a inhibition studies in Nalm6 cell line.
Indeed, G9a/GLP coactivator function is implicated in the upregulation of GC mediated genes
causing GC mediated lymphoblast death.
First, we performed a preliminary protein expression analysis on endogenous Aurora
Kinase B regulators in Pre B697 and R3F9 cell lines. Pre B697 cell line is a GC- sensitive B-ALL cell
line, while R3F9 which is derived from Pre B697 cell line is a GC-resistant cell line. We wanted to
explore the basic possibility of Aurora Kinase B regulators being implicated in GC- resistance in
R3F9 cell line. Interestingly Eb1 is slightly overexpressed in R3F9 cell line (Figure 4, panel 4)
compared to Pre B697 cell line which might implicate it in GC- resistance.
15
Figure 4: Eb1 is overexpressed in R3F9 cell line
Pre B697 cell line is a model of B-ALL which is sensitive to GCs or dexamethasone induced cell
death. R3F9 cell line is resistant to GC action. Lysates of both cell lines were immunoblotted for Chk1, Eb1,
PP1A and PP2A. n – number of independent experiments.
Using literature, we identified Calyculin A as a potential inhibitor of PP2a and tested
varying concentrations of both the drugs for 30 minutes of treatment time as authors described.
We performed Immunoblotting with phosphorylated LATS1 (pLATS1) and phospho-threonine
(pThr) antibodies on the lysates after inhibitor treatments. pLATS1 is a known substrate of PP2a
[33]
and hence it is used as a specific marker for the PP2a inhibition experiments. Inhibition of
PP2a is expected to raise the level of pLATS1 compared to control DMSO. We also used a general
pThr marker, as PP2a is known to have a lot of substrates and its inhibition would lead to rise in
general phospho-threonine levels of many proteins compared to control DMSO. We show that
treatment with Calyculin A increases the level of pLATS1 and pThr (Figure 5a and 5b) which means
it is successfully inhibiting PP2A activity and we chose 10nM as the ideal concentration for our
subsequent inhibition studies, since inhibitor treatments in our experiments would be much
longer (6hrs).
16
Figure 5: Inhibitor Selection and Optimization
Nalm6 cells were treated for 30min with different concentrations of the PP2a inhibitor Calyculin A
(Caly) to assess its effectiveness. pLATS1 was chosen as a specific substrate of PP2a and total protein with
phospho-Threonine (p-Thr) was chosen as another general substrate of PP2a to test the action of inhibitor.
After treatment with inhibitor, Lysates were immunoblotted with pLATS1 and P-Thr antibodies and actin
antibody as a loading control.
Then we wanted to check whether PP2a inhibition would mediate any direct changes in
the expression levels of proteins of interest. After inhibitor treatments for 6hrs, we performed
Western blots on the lysates for G9a, GLP, Aurora Kinase B (AurKB) and PP2a. We observed that
over three independent experiments, G9a/GLP and AurKB are being overexpressed when PP2a
has been inhibited for 6hrs with 10nM of Calyculin A with respect to control DMSO (Figure 6a).
This shows that investigating G9a phosphorylation levels in Nalm6 will be tricky, since the basal
expression of proteins in both situations are vastly different when treated with PP2A inhibitor.
17
Figure 6: PP2a Inhibition with 10nM inhibitor causes overexpression of G9a/GLP and AurKB
Nalm6 cells were treated with 10nM Calyculin A for 6 hours. Lysates were then immunoblotted for
G9a/GLP, Aurora Kinase B (AurKB) and PP2a for analysis (a) and, also pLATS1 (a) and P-Thr (b) as inhibitor
treatment controls. Tubulin was used as a loading control. n – number of independent experiments.
PP2A Inhibition changes expression profile of GC target genes
Along with Protein analysis, we wanted to analyze the changes in G9a/GLP coactivator
functions by observing the expression patterns of dex-regulated genes which are activated by
G9a/GLP. Since PP2A is known to deactivate Aurora Kinase B, we hypothesize that its knockdown
would lead to higher activity of Aurora Kinase B and more G9a phosphorylation, thus inhibiting
its coactivator function. So, we would expect to see lesser induction of G9a dependent genes
when PP2A is inhibited. We chose GILZ, NFKBIA, TXNIP and GPR56 as ideal candidates for analysis
since these genes which are dependent on G9a/GLP are also involved the GC-induced death of
B-ALL cells. We also chose a control gene FKBP5 which is independent of G9a/GLP but regulated
by dex. For achieving our goals, we chose to treat cells with inhibitor and dexamethasone
together for 6hrs followed by isolation of mRNA and performing qRT-PCR on the genes of
interest. Over three independent experiments, we observed a statistically significant decrease in
the expression of GPR56 and FKBP5 genes (Figure 7) by dex, with complete abolishment of
induction of GPR56 when treated with 10nM of Calyculin A compared to control DMSO. Although
not statistically significant, we observed a slight decrease in the expression of GILZ (Figure 7).
Since FKBP5 is an independent gene, we would not expect to find any changes in its expression
when PP2A is inhibited. Since we observed changes even in the FKBP5 gene, we can safely say
18
that 10nM concentration of inhibitor might be causing non-specific effects and steps should be
taken to reduce it.
Figure 7: PP2a inhibition with 10nM inhibitor causes changes in both G9a/GLP dependent and
independent gene expression pattern
Nalm6 cells were treated with 10nM Calyculin A and dexamethasone together for 6 hours
followed by RNA extraction and cDNA preparation. The expression profile of genes of interest: GILZ,
NFKBIA, GPR56, TXNIP (G9a/GLP dependent) and FKBP5 (G9a/GLP independent) was analyzed by real time
quantitative PCR (qPCR) and normalized to Actin mRNA expression. n-number of independent experiments
Approach to resolve the side effects of PP2A Inhibition
PP2a is known to play an important role in mitosis and this raised doubts to whether its
inhibition is causing changes in the cell cycle progression. This is an important factor since
G9a/GLP performs and regulates their coactivator functions in G1 phase of cell cycle. Also, their
expression patterns are different with each phase of cell cycle with maximal expression in G1
phase. Since we saw changes in induction of FKBP5 gene which is independent of G9a/GLP (Figure
7) and should ideally be non-specific to PP2A inhibition, this amplified our doubts, making us
investigate the effects on cell cycle progression. Thus, we performed Cell sorting assay using flow
cytometry (FACS) with Propidium Iodide staining and observed that 10nM concentration of
Calyculin A is inhibiting cell cycle progression and cells are stacking in G2/M phase (Figure 8a,
highlighted in red color). To mitigate that, we decided to use lower concentrations of inhibitor.
Using pLATS1 and pThr as substrates for optimization experiments and simultaneous cell sorting
analyses, we have chosen 3nM concentration as the lowest possible concentration for
subsequent testing as it is successfully inhibiting PP2A due to rise in pLATS1 and pThr levels
19
(Figure 9a and 9b) and cells are being stacked in G1 phase of the cell cycle (Figure 8d, highlighted
in green color).
Figure 8: Problems with PP2a Inhibition and Solution to it
Nalm6 cells were treated with Calyculin A of different concentrations and the cells were
collected and fixed with Ethanol and PBS. They were then labeled with Propidium Iodide and sorted with
Flow Cytometry for cell cycle analysis. (a) - Percentage of cells in each phase of cell cycle when treated with
Calyculin 10nM, control DMSO and non-treated cells with or without dexamethasone for 6 hours. (b) and
(c) – represents the raw data obtained from Flow Cytometry of DMSO and Calyculin 3nM treated Nalm6
cells for 6 hours respectively. (d) - Raw counts of (b) and (c) along with control DMSO and other
concentrations of inhibitor are converted into percentages using software.
20
Figure 9: 3nM Calyculin A is the minimum concentration which is causing an effect
Lower concentrations of Calyculin A were tested (1nM, 2nM, 3nM, 4nM, 5nM) to assess their
effectiveness in Nalm6 cells. pLATS1 was chosen as a specific substrate of PP2a and phospho-Threonine
(P-Thr) was chosen as another general substrate of PP2a to test the action of inhibitor. Nalm6 cells were
treated with the inhibitor for 6 hours. Lysates were then immunoblotted for pLATS1 and P-Thr levels.
Protein and Gene expression Analyses with modified Inhibitor treatment
a. PP2A Inhibition causes overexpression of G9a/GLP
With the same hypotheses for the previous experiments (Figure 6 and Figure 7) on our
mind, we performed a single round of experiments with 3nM Calyculin A to analyze the changes
in expression levels of proteins of interest due to PP2A inhibition. After 6hr treatment, we
observed that G9a/GLP are still upregulated even with lower concentration of inhibitor whereas
Aurora Kinase B was downregulated (Figure 10).
21
Figure 10: PP2a inhibition with 3nM drug causes overexpression of G9a/GLP
Nalm6 cells were treated with 3nM Calyculin A for 6 hours. Lysates were then immunoblotted for
G9a/GLP, Aurora Kinase B (AurKB) and PP2a for analysis. n-number of independent experiments
b. PP2A Inhibition specifically changes profile of G9a/GLP dependent genes
Similarly, we repeated the experiments for gene expression analysis with 3nM
concentration of Calyculin A. We performed qRT-PCR and observed changes in the gene
expression profile for a single independent experiment. Interestingly GPR56 was not induced at
all just like the previous experiments (Figure 7 and Figure 11). There was no change in the
expression of FKBP5, thus showing that PP2a inhibition with 3nM Calyculin A is more specific to
G9a/GLP dependent genes (Figure 11). There are also observable changes in NFKBIA, TXNIP and
GILZ (Figure 11). More replicates are needed to reach a significant conclusion.
22
Figure 11: PP2a inhibition with 3nM drug causes changes in G9a/GLP dependent gene expression pattern
Nalm6 cells were treated with 3nM Calyculin A and dexamethasone together for 6 hours. RNA is
extracted and cDNA is prepared. The expression profile of genes of interest: GILZ, NFKBIA, GPR56, TXNIP
(G9a/GLP dependent) and FKBP5 (G9a/GLP independent) is analyzed by real time quantitative PCR (qPCR)
and normalized to Actin mRNA expression. n-number of independent experiments
DISCUSSION
PP2A Exhibits an Indirect effect on G9a Phosphorylation in Cos7
B-ALL is a type of leukemia commonly found in children where too many precursor B
lymphocytes are found in blood and bone marrow. Glucocorticoids (GCs) are known to be an
effective mode of treatment for remission of B-ALL. But relapsed tumors are found to be resistant
to GC action. G9a/GLP are euchromatic histone lysine methyl transferases which exhibit
coactivator function for GC regulated genes. Auto-methylation of G9a/GLP is responsible for its
coactivator function whereas G9a/GLP phosphorylation by Aurora Kinase B inhibits its coactivator
function. The coactivator function is crucial in activating the genes causing GC-induced cell death
in B-ALL cells. Since PP2A deactivates Aurora Kinase B
[25][26]
, we hypothesized that PP2A
knockdown would lead to more activation of Aurora Kinase B, thus leading to more G9a
Phosphorylation by Aurora Kinase B
[2][4]
. But we observed the opposite in Cos7; that is lesser G9a
phosphorylation when PP2A is knocked down (Figure 2). This is interesting as it shows that the
phosphatase activity of PP2A is not directly involved by affecting the activity of Aurora Kinase B,
rather the decrease in G9a phosphorylation is an indirect effect of its knockdown. Thus, we
speculate that knockdown of PP2A might alter the activity of an unknown target which has a
downstream effect on G9a phosphorylation significantly.
23
Chk1 has an impact on G9a stability
Interestingly, what can be considered as a novel observation, we observed that whenever
siChk1 and HA tagged G9a plasmid were co-transfected, there was almost always no
overexpression of HA-G9a compared to controls and knockdowns of other proteins. This was
significantly held true for three independent experiments (Figure 3). There were two possible
explanations for this, either issues in co-transfection of siChk1 and HA-G9a specifically or Chk1
being implicated in the stability of G9a. To confirm our suspicions, we conducted another
experiment to assess the effects of Chk1 knockdown on endogenous G9a along with
overexpressed G9a. Unexpectedly, we observed that knockdown of Chk1 also destabilizes
endogenous G9a along with overexpressed G9a, thus confirming our hypothesis of Chk1 having
an impact on G9a stability. Therefore, we speculate that Chk1 might have impact on stability of
G9a either directly by protein-protein interactions or indirectly by its effect on cell cycle control
and progression. Chk1 is known to play a major role in cell cycle, with its functions reported to
be crucial in DNA repair during replication, S phase and G2/M transition, along with other
functions in M phase. Thus, when Chk1 is knocked down, we speculate that it may alter the profile
of cells in different stages of cell cycle and it might result in overall changes to the expression of
G9a.
Inhibition of PP2A leads to overexpression of G9a/GLP in Nalm6
After deducing that PP2A exhibits an effect on G9a phosphorylation in Cos7, we selected
it as the prime candidate for the subsequent studies in Nalm6, a physiological model of B-ALL. It
is important to remember that, the classical mechanism of Aurora Kinase B regulation discussed
in this paper holds true for most of the cells in normal tissues. Interestingly, the mechanism of
regulation of Aurora Kinase B has not been characterized in B lymphocytes. We expect that the
mechanism of regulation of Aurora Kinase B in B lymphocytes, may or may not be different from
the model discussed in this paper since there is a report of a slightly different mechanism in T
lymphocytes
[32]
, which come from the same Lymphoid lineage as B cells. In addition, Aurora
Kinase B which is only significantly expressed during mitosis in most cells, is also reported to be
fairly expressed in the G1 phase of cell cycle in T-cells
[32]
. This suggests that there might be some
differences between lymphocytes and other cells regarding Aurora Kinase B expression and
regulation. Thus, we are also interested to see whether the model for regulation of Aurora Kinase
B discussed in this paper is relevant to any degree in B-ALL cells.
Firstly, we wanted to observe whether PP2A inhibition would have any direct effect on
the proteins of interest (G9a/GLP and AurKB) in Nalm6 cells. We observed that when PP2A was
inhibited with either 10nM or 3nM Calyculin A inhibitor, G9a/GLP proteins were overexpressed.
Initially when we observed overexpression of G9a/GLP when treated with 10nM inhibitor, we
speculated that it might be due to significant changes in cell cycle progression since PP2A is an
important cell cycle regulator. In fact, we observed that cells were stacking in G2/M phase of the
24
cell cycle when treated with 10nM inhibitor. Hence to reduce this side effect, we chose to work
with 3nM concentration of the inhibitor, which was causing the cells to stack in G1 phase. Even
when treated with lesser concentration of inhibitor we observed that G9a/GLP were being
overexpressed. Thus, we speculate that, when PP2A is inhibited, it leads to more overall
phosphorylation of G9a/GLP and these changes in the Post translation modifications might be
leading to lesser turnover or degradation of proteins. Interestingly, we clearly observed a shift in
G9a/GLP bands in the Western Blot when PP2A was inhibited with 10nM inhibitor, showing that
they might be hyper-phosphorylated. But this is in stark contrast with the results in Cos7 cells,
where PP2A knockdown was causing lesser G9a phosphorylation. We speculated that this
discrepancy may be due to the fundamental differences between the cell types and cell biology
of Cos7 and Nalm6.
We also observed different changes in the expression of Aurora Kinase B when treated
with 10nM and 3nM inhibitor. Aurora Kinase B was overexpressed when PP2A was inhibited by
10nM inhibitor and slightly down-regulated when PP2A was inhibited by 3nM Inhibitor. The
possible explanation for this phenomenon is the changes in cell cycle progression. As mentioned
before, when PP2A was inhibited by 10nM inhibitor, the cells were stacked in G2/M phase.
Aurora Kinase B is strongly expressed in M phase of the cell cycle and thus stacking of cells in
G2/M can lead to relative overexpression of Aurora Kinase B compared to control due to more
cells being present in G2/M phase. On the contrary, when treated with 3nM inhibitor, cells were
stacking in G1 phase of the cell cycle and Aurora Kinase B is weakly expressed in G1 phase
compared to M phase in lymphocytes. This leads to less significant changes in the expression of
Aurora Kinase B when treated with 3nM inhibitor.
Inhibition of PP2A affects the coactivator function of G9a/GLP
Since the coactivator function of G9a/GLP is important in activating genes causing GC-
induced cell death in Nalm6 cells, we wanted to observe the changes in the coactivator function
of G9a/GLP when PP2A is inhibited in Nalm6 through changes in the induction of G9a/GLP
dependent genes. Since the coactivator function of G9a/GLP is only specific to the G9a/GLP
dependent GR target genes, we expect that PP2A inhibition would only cause any changes in the
profile of dependent genes but not in independent genes. Initially when we treated with 10nM
inhibitor, we observed changes in the induction of independent genes (FKBP5) along with some
dependent genes (GPR56 and GILZ) which is not as expected. We speculated that this is due to
the side effects of PP2A inhibition with 10nM concentration of drug. Since PP2A has large number
of substrates, its inhibition might be causing changes in some other substrates of PP2A which can
affect the induction of the independent gene FKBP5. In addition, 10nM concentration of the
inhibitor might be too potent, causing more non-specific and side effects, in addition Calyculin A
also inhibits PP1A along with PP2A, which reinforces this speculation since PP1A acts similarly to
PP2A and has many substrates which might affect the induction of FKBP5.
25
As we previously discussed, we conducted the subsequent experiments with 3nM
concentration of inhibitor to reduce the effects on the cell cycle progression. 3nM was the lowest
concentration we tested at which the drug was being effective. This diminishes the possibility of
non-specific and side effects of the drug. Indeed, we observed changes in induction of only
dependent genes, whereas the independent gene FKBP5 was unaffected when treated with 3nM
inhibitor showing that it was being more specific. Initially our hypothesis was, since PP2A is
known to deactivate aurora kinase B according to the model of regulation of Aurora Kinase B we
discussed in this paper, its inhibition would lead to more activity of Aurora Kinase B and more
G9a Phosphorylation. This would lead to reduced coactivator function of G9a/GLP and thus less
induction of G9a/GLP dependent genes. Interestingly, out of the 4 dependent genes, three of
them (GPR56, NFKBIA, TXNIP) were less induced whereas one was more induced when compared
to control, thus largely agreeing with our hypothesis. But this is in stark contrast to our
observation of changes in G9a phosphorylation in Cos7 cells. In the cos7, we observed that PP2A
knockdown would cause lesser G9a phosphorylation and this would lead to an opposite
hypothesis where PP2A inhibition should lead to more co-activator function. This discrepancy
between the observations in Cos7 and Nalm6 might be due to the inherent differences in the cell
types and cell biology and the mechanistic differences between silencing RNA and the action of
an inhibitor.
Future Direction
In the future, experiments can be conducted to directly analyze the changes in G9a
phosphorylation in Nalm6 cells, when PP2A is inhibited. It might not be very suitable since we
already showed that PP2A inhibition causes overexpression of G9a/GLP. If the basal expression
levels of G9a/GLP are vastly different, it can be difficult to compare the G9a phosphorylation
levels. Cell Death assays can also be conducted to assess the viability and sensitivity of cells to
dexamethasone treatment when PP2A is inhibited, since our final goal is to enhance the GC
treatment of B-ALL cells. An alternative approach may also be taken for inhibiting PP2A, by
serum starving the cells in G1 phase and knocking down PP2A either by a conditional knockout
or by an inhibitor, diminishing the problems with cell cycle progression. Finally, it would be
worth investigating the effect of Chk1 on G9a stability and deducing the model for interactions
between Chk1 and G9a.
MATERIALS AND METHODS
Cell Culture and Inhibitor Treatment
For Cos7 cells, DMEM (Dulbecco’s Modification of Eagle’s Medium) with 4.5 g/L Glucose (high
concentration) and 10% Fetal Bovine Serum (FBS) was used for culture and maintenance. For
experimental setups, 10 cm dishes were used and the cells were plated with a seeding density of
either 1.3*10
6
cells or 1.5*10
6
cells for Transfection.
26
For Nalm6 cells, RPMI-1640 (Gibco) media with 10% FBS was used for culture and maintenance.
At least a cell viability of 90% was preferred for maintenance and experimental considerations.
One week after media was freshly prepared, Glutamax was added to the media to replenish the
amino acid Glutamine. For experimental setups, 6 well plates were used and cells were plated
with a concentration of 0.5*10
6
cells/ml for each well.
Inhibitor treatments were performed around 24hrs after culturing Nalm6 in 6 well plates.
Calculated volumes of Calyculin A (inhibitor for PP2A) was added to the wells to obtain the testing
concentrations of 3nM and 10Nm for Protein analyses with a treatment time of 6hrs. Nalm6 were
treated simultaneously with 3nM or 10Nm Calyculin A and 100nM dexamethasone for RNA
expression analyses for 6hrs. The volume of control DMSO added was equivalent to the highest
volume of the inhibitor added in a set of samples.
Protein knockdown with siRNA and Plasmid overexpression
Transfection (or Co-transfection) was performed on Cos7 cells around 24 hours after the 10cm
dishes were plated. We made sure that the dishes were 60-70% confluent with cells spread
evenly across the surface of the dish. 20ul of siRNA (at 20uM) specific for each of the Aurora
Kinase B regulators Chk1, Eb1, PP1a and PP2a and a non-specific SiNS were either transfected
alone for knockdown validation studies or co-transfected with 5ug of plasmid HA-G9a FL WT (HA
tagged G9a Full Length Wild Type) per 10cm dish for analyzing G9a phosphorylation. The siRNA
was packaged with lipofectamine siRNAi max (Invitrogen) and the plasmid HA-G9a FL WT with
Lipofectamine 2000 (Invitrogen). All the reagents were prepared in Opti-MEM media (Gibco).
Exactly at 6 hours after transfection reagents were added, 2mL of DMEM with 4.5 g/L Glucose
was added. Plates were harvested on the third day after transfection.
Cell Lysis, Protein Extraction and Dosage
For Cos7 cells, the 10cm dishes were washed three times with 1X DPBS (Dulbecco’s Phosphate
Buffered Saline). Required amount of RIPA mix was prepared by mixing the Protease Inhibitors
βGP (5ul/ml), Na 3VO 4 (5ul/ml) and 25X (40ul/ml) in the RIPA buffer. Around 400 – 600ul of RIPA
mix was added to each 10cm dish depending on the number of cells alive in the plate and
incubated at 4
0
C for at least 20min. The cell extracts would then be scraped, collected and then
centrifuged @ 12500 rpm for 10min at 4
0
C. The supernatant was then collected to obtain the
protein extracts.
For Nalm6 cells (suspension), they were first centrifuged and then the pellet was washed with 1X
DPBS once. Required amount of RIPA mix was prepared with the exact same composition as
mentioned above. Around 150-250ul of RIPA mix was added to each pellet and then incubated
at 4
0
C for at least 20min followed by centrifugation @ 12500 rpm for 10min at 4
0
C. The
supernatant was then collected to obtain the protein extracts.
27
Once the proteins were extracted from both types of cells, their concentrations were quantified
using Bradford Assay technique. 5X Protein Assay Concentrate (Biorad) was used for sample
preparation and the instrument UV-Spectrophotometer (Biorad) was used according to the
manufacturer’s protocols. Bovine Serum Albumin (BSA) (1mg/ml) was used for standard
preparation.
Immunoprecipitation (IP)
All the immunoprecipitation experiments were performed with phospho-threonine
(abcam) Antibody. A minimum of 700ug of protein was required, but 1mg of protein was the
preferred amount. RIPA buffer was added to the protein extract to make the total volume of the
sample as 1ml. Then the Protease inhibitors βGP (5ul), Na 3VO 4 (5ul) and 25X (40ul) were added
to the sample. 5ul of phospho-threonine antibody was then added to the sample and incubated
at 4
0
C overnight under agitation.
The next day (at around 12-16hrs after incubation) 80ul of Agarose A/G Beads was taken
and activated by washing with RIPA 2 times. The beads were then resuspended with some of the
sample and then transferred back into the remaining sample. The samples with beads were
incubated at 4
0
C for 2hrs under agitation.
After 2hrs, the samples were centrifuged and the pellets (beads) were washed with RIPA
buffer for three times. After the final wash and removal of the supernatant, 70ul of 1x of Laemmli
(L1X) was added to the beads and heated @ 90
0
C for 5min. After heating, the beads were then
centrifuged @10,000 rpm for 1min. This supernatant consists of the immunoprecipitated protein
and was loaded onto gels for SDS-PAGE and Immunoblots.
SDS-PAGE and Immunoblots
SDS-PAGE consists of resolving (6-10% depending on proteins to be visualized) and stacking gels
(6%). Resolving gels were made with distilled water, 30% acrylamide mix, 1.5M Tris (pH 8.8), 10%
SDS, 10% ammonium persulfate and TEMED. 6% stacking gels were made with distilled water,
30% acrylamide mix, 1.5M Tris (pH 6.8), 10% SDS, 10% ammonium persulfate and TEMED. The
volumes of each component of the gels depends on the percentage of the gel. After solidified,
these gels were setup for Gel electrophoresis and filled with Running buffer (1X Tris-Glycine-SDS).
30ug of protein samples were mixed with 10ul of 2x Laemmli (L2X) and then heated at 90
0
C for
5min. The samples were then loaded onto the gels and electrophoresis was performed.
Once finished, the proteins in the resolving gels were then transferred to a PVDF
membrane (Biorad) by making a transfer sandwich with blotting papers, PVDF membrane and
resolving gels in transfer buffer (3:1:1 - Distilled Water: 5X Turbo Transfer Buffer (Biorad): 100%
Ethanol). The membranes were then transferred into the blocking buffer which consisted of 5%
dried milk in 1X Tris Buffered Saline containing 0.1% Tween (1X TBST). The membranes were
blocked for at least 30min. Then the membranes were split into required compartments and
incubated with primary antibodies for proteins of interest for overnight at 4
0
C.
28
On the next day, membranes were first washed with 1X TBST three times for a total of
30min. The membranes were then incubated with HRP-conjugated secondary antibodies
(1/10,000 concentration) for 1hr at room temperature. Then the membranes were washed again
with 1X TBST for three times. The membranes were then incubated with ECL
TM
Prime Western
Blotting Detection Reagent (GE Healthcare) and detected by Image Lab software.
Primary Antibodies Dilution Species Supplier Reference
β-Actin 1/5000 Mouse Sigma A5441
G9a 1/1000 Rabbit Sigma G6919
GLP 1/1000 Rabbit Millipore 09-078
Aurora Kinase B 1/1000 Mouse abcam ab2254
Chk1 1/1000 Mouse Cell Signaling 2360
PP1a 1/1000 Rabbit Cell Signaling 2582
PP2a 1/1000 Rabbit Cell Signaling 2259
Eb1 1/1000 Mouse Cell Signaling 2164
HA 1/1000 Rat Roche 3F10
β-Tubulin 1/5000 Mouse Cell Signaling 2146
phospho-Threonine Rabbit Millipore AB1607
pLATS1 1/1000 Rabbit Cell Signaling 8654
GAPDH 1/10000 Rabbit Sigma G9545
RNA Extraction and cDNA Preparation
For Cos7 cells (adherent), they were washed once with DPBS. Then Trizol (Ambion) is added into
the 10cm dishes and then the cells were scraped to collect the Trizol extracts. For Nalm6 cells
(suspension), Trizol is directly added to the DPBS washed cells and suspended well. The trizol
extracts were stored at -80
0
C.
Trizol extracts were then defrosted and then worked with Direct-Zol
TM
RNA prep kit (Zymo
Research) to extract the total RNA according to manufacturer’s protocols. The purity and
concentration of RNA is measured using Nano-Drop 2000 instrument (Thermo Fisher Scientific)
according to the manufacturer’s instructions. RNA was then stored at -80
0
C.
cDNA was prepared using Superscript III (Invitrogen) kit using 1000ng or 1ug of RNA. The
cDNA is diluted to 1:10 volume with qPCR water (Roche) for usage in real-time PCR. Standard
cDNA is then prepared by serial dilutions.
Real-Time PCR (qPCR)
qPCR was performed using SYBR-Green I Master mix kit (Roche) and specific primers to the genes
of interest on Lightcycler 480 instrument (Roche) according to the manufacturer’s protocols.
29
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Abstract (if available)
Abstract
Glucocorticoid hormones are known to activate cellular responses through GR (Glucocorticoid Receptor) mediated transcriptional regulation of target genes. After hormone stimulation, GR binds to GBR (GR Binding Regions) in the Genome and subsequently recruits coregulators which can either stimulate or inhibit the transcription in a gene specific manner. Our focus is on the coregulators, H3K9 Histone Methyl-Transferases G9a and GLP (G9a Like Protein). G9a/GLP can act gene specifically as coactivator or corepressor. Indeed, G9a/GLP methylates H3K9, a well-known repressive mark. Dr. Stallcup’s laboratory demonstrated that G9a/GLP can also act as coactivator of GR. This coactivator function is controlled by a Post Translational Modification Switch in their N-Terminal Transcriptional Activation Domains. Auto-Methylation at a specific Lysine in that domain enables its coactivator function
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Adusumilli, Sushanth
(author)
Core Title
Regulation of Aurora kinase B and its effect on phosphorylation of G9a/GLP
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
08/08/2018
Defense Date
05/08/2018
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Aurora kinase B,coregulator,G9a,glucocorticoid receptor,leukemia,OAI-PMH Harvest,phosphorylation,regulation
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application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Stallcup, Michael R. (
committee chair
), An, Woojin (
committee member
), Xu, Jian (
committee member
)
Creator Email
sadusumi@usc.edu,sushanthadusumilli@gmail.com
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https://doi.org/10.25549/usctheses-c89-61560
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UC11671705
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etd-Adusumilli-6693.pdf (filename),usctheses-c89-61560 (legacy record id)
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etd-Adusumilli-6693.pdf
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61560
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Thesis
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Adusumilli, Sushanth
Type
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University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
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The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
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Tags
Aurora kinase B
coregulator
G9a
glucocorticoid receptor
leukemia
phosphorylation
regulation