The Role of Gab2 Phosphorylation Sites in Hematopoietic Signaling

THE ROLE OF GAB2 PHOSPHORYLATION SITES IN HEMATOPOIETIC SIGNALING

SHEETAL VERMA

Submitted in partial fulfillment of the requirements For the degree of Masters of Science

Thesis Advisor: Dr. Kevin D. Bunting

Department of Biology CASE WESTERN RESERVE UNIVERSITY

May, 2010

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______Sheetal Verma______candidate for the ______MS____degree *.

(signed) Dr. Michael Benard (chair of the committee)

Dr. Kevin Bunting Dr. Arnold Caplan Dr. Stephen Haynesworth Dr.Cheng-Kui Qu

(date) ______1/29/2010______

*We also certify that written approval has been obtained for any proprietary material contained therein.

DEDICATED

TO MY BELOVED MA AND PAPA

TABLE OF CONTENTS

1. Introduction 1.1 The Gab Family of Proteins 6 1.2 Discovery and Structural Similarities 6 1.3 Role in Cytokine Signaling and Hematopoiesis 8 1.4 Potential Role in Cancer 11

2. Materials and Methods 2.1 Purification and Sequencing of the Plasmid 14 2.2 Generation of Producer Lines and Measurement 14 of Transduction Efficiency 2.3 Transduction of Ba/F3 16 2.4 Validation of Gab2 Mutants 17 2.5 Dominant Negative Effects of Mutants 18 2.6 Functional Impact of Mutants 19

3. Results 3.1 The Gab2 Mutants Show Binding Defect Toward 20 Their Respective Partners 3.2 The Presence of the Mutants Has Mild Effect on Binding of Endogenous Gab2 21 3.3 Gab2 WT cells have No Growth Advantage Over the Mutants, but These Cells show higher Cell Viability 22

4. Discussion 24

Bibliography 35

LIST OF FIGURES

Figure 1: Gab2 interactions with binding partners 29

Figure 2: Uniform Gab2 expression levels seen in Ba/F3 cells expressing WT, G2/SHP-2 binding site mutant (Gab2Y604F/Y633F) and G2/p85 binding site mutant (Gab2Y441F/Y465F/Y574) 30

Figure 3: Transduction of Ba/F3 cells with mutated Gab2 constructs results in defective binding of Gab2 with SHP2 and p85 30

Figure 5: Ba/F3 Gab2WT cells show no growth advantage over Gab2/SHP2 and G2/p85 mutant samples, after cytokine withdrawl and subsequent IL3 stimulation 32

Figure 6: Ba/F3 Gab2 cells show significantly higher cell viability compared to mutant samples, after IL3 stimulation 33

LIST OF TABLES

Table 1 Densitometric analysis of Gab2, G2/SHP2 and G2/p85 reveals a mild defect in interaction of total Gab2 with its respective partner when over-expressing G2/p85 mutant, but not G2/SHP2 mutant 34

ACKNOWLEDGEMENTS

I want to extend my sincere thanks to my advisor and mentor Dr. Kevin Bunting for his crucial guidance and patient review of my work. I would also thank my advisory committee members Dr. Arnold Caplan, Dr. Stephen Haynesworth and Dr. C K Qu for lending me their expert views and precious time.

I would take this opportunity to express my gratitude to the members of Bunting lab, Dr. Kristy Miskimen, Dr. Zhengqi Wang and Dr. Greg Li for supervising and helping me throughout the length of this project.

I am also grateful to the Biology department at Case Western Reserve University and also to the Flow Cytometry Core of the Case Cancer Center.

Last, but certainly not the least, I extend my gratefulness to my friends and family for their never-ending love and support.

LIST OF ABBREVIATIONS

PI-3K Phosphoinositide 3-kinase

MAPK Mitogen-activated protein kinase

ERK Extracellular signal-regulated kinase

MSCV Murine Stem Cell Virus

EDTA Ethylenediaminetetraacetic acid

SDS Sodium dodecyl sulfate

PAGE Polyacrylamide gel electrophoresis

WT Wild Type

IL Interleukin

LB Lysogeny Broth

TE Tris-EDTA

FBS Fetal Bovine Serum

CS Calf Serum

GFP Green Fluorescent Protein

DMEM Dulbecco's Modified Eagle Medium

IMDM Iscove's Modified Dulbecco's Medium RPMI-1640 Roswell Park Memorial Institute medium O/N Over Night

PEI Polyethyleimine

CM Conditioned media

FACS Fluorescence-activated cell sorting

PVDF Poly vinylidene fluoride

The Role of Gab2 Phosphorylation Sites in Hematopoietic Signaling

Abstract

SHEETAL VERMA

The Grb-2 Associated Binder (Gab) family of proteins has been known to play significant roles in cytokine and receptor signaling. Delineating the precise mechanism of Gab- mediated signaling is critical to understanding how cytokines, growth factors and oncoproteins regulate biological functions of cell growth and differentiation. Recent approaches in protein biochemistry and systems biology have revealed how Gab proteins are subject to complex regulation by phosphorylation events as well as protein-protein interactions. The Gab proteins possess structural motifs for their receptor recruitment and are also involved in transduction of receptor-derived signals. These proteins can be negatively regulated by protein phosphorylation that causes alterations in their ability to interact with crucial interaction partners or in their sub-cellular localization. Gab2 which is known to interact with many common receptors through its various signaling domains was chosen for this study and its interaction with two of its most crucial partners, SHP2 and p85, was validated. It was biochemically confirmed how point mutations on Gab2 disrupt its binding with these partners and also effect cell growth and proliferation. This led to a better understanding of the critical role that Gab2 plays in hematopoietic signaling.

1. INTRODUCTION

1.1 The Gab Family of Proteins

The Gab proteins, Gab1, Gab2 and Gab3, comprise a family of scaffolding/docking

molecules involved in multiple signaling pathways mediated by receptor tyrosine kinases

(RTKs) and non-RTK receptors. Gab proteins integrate and amplify signals from a wide

variety of sources including growth factor, cytokine and antigen receptors as well as cell

adhesion molecules. They also contribute to signal diversification by channeling the

information from activated receptors into signaling pathways. They are subject to

complex regulation by feed-forward and feedback phosphorylation events as well as

protein-protein interactions. These proteins range from 50 to 100 kDa in size (Gu and

Neel, 2003). The Gab molecules were, originally identified as the mammalian homologs

of the Daughter of Sevenless (DOS) Drosophila adapter proteins. They also display

sequence similarity to Suppressor of Clear 1 (Soc1), which was identified by genetic

screens in C. elegans (Herbst et. al., 1996; Schutzman et. al., 2001). Now with

biochemical and structural studies revealing their true mechanism of action, it has

become evident that Gab proteins are crucial signaling elements and are key regulators

and mediators in many biological activities

1.2 Discovery and Structural Similarities

Gab1 was originally identified as a binding protein for Grb-2 (Holgado-Madruga et. al.,

1996) and Gab2 was isolated by the purification of a binding partner for SHP2 (Gu et.al.,

1998). The discovery of Gab3 was aided by a large sequencing project and its isolation

was based on sequence similarities to Gab1 and Gab2 (Wolf et. al., 2002). Very recent

entries at both the genomic DNA and transcript level have been recorded for Gab4 gene in both humans and chimpanzees, but not mice. The human Gab4 gene is located on chromosome 22q11.1 and its nucleotide sequence is most related to Gab2 (Wöhrle et. al.,

2009).

Both Gab1 and Gab2 are expressed ubiquitously with high levels seen in brain, kidney, lung, heart and ovary (Holgado-Madruga et. al., 1996; Gu et. al., 1998 and Zhao et. al.,

1999). For Gab3, hematopoietic tissue has the highest expression. The overall sequence homology between Gab family members is about 40-50%. All Gab proteins share a similar modular structure, including a Pleckstrin Homology (PH) domain at their N- terminus, proline-rich regions in the central part and multiple phosphorylated tyrosine residues. The PH domain is an approximately 100 amino acid domain that binds phosphoinositides. The PH domain is a similar structural fold abundant in signaling molecules such as Ras-GTPase Activating Protein (Ras-GAP), Phospholipase C-gamma

1 (PLC-γ1), β- spectin, Akt and many others. Gab2 binds preferentially to the PI-3K product phosphatidylinositol-3,4,5-trisphosphate (PIP3), which is only found within the plasma membrane( Zhao et. al.,1999). The PH domain mediates recruitment of Gab2 to phagocytic cups induced by FcγRI and is required for fibroblast growth factor-induced tyrosine phosphorylation of this docking protein. The PH domain might play an important role to localize or to concentrate Gab proteins to membrane areas where receptors are activated (Rabbe et.al., 1996).

The phosphotyrosine domains and the proline-rich sequences (PXXP) are potential binding sites for Src Homology2 (SH2) and SH3 domains, respectively. The positions of the SH2-binding tyrosine-based motifs and the SH3-binding proline-rich sequence are

conserved among the Gabs. These are one of the most prominent motifs in signaling molecules due to their relevance in binding and ‘docking’ phosphorylated tyrosine residues or directing protein-protein interaction ( Gu and Neel, 2003; Rohrschneider et. al.,2002).

1.3 Role in Cytokine Signaling and Hematopoiesis

A fundamental mechanism for regulation of Gab-mediated signal transduction is site- specific tyrosine phosphorylation of these proteins. Their role in regulation of cytokine signaling pathways is pivotal to understanding the dysregulation in hematologic disease.

Figure 1 shows the Gabs are especially involved PI-3K and MAPK pathways, which has also been described by Yart et. al., 2001; Gu, 2003 and Rohrschneider,2000. Many of the ligands and receptors associated with Gab activation have now been defined. Gabl is tyrosine-phosphorylated in response to insulin, epidermal growth factor (EGF), platelet- derived growth factor (PDGF), stem cell factor (SCF), thrombopoietin (TPO), IL-3, erythropoietin (EPO), and the stimulation of gp 130 and by the receptors of the IL-6- family cytokines (Nishida,2003). Gab2 is tyrosine-phosphorylated in response to IL-2,

IL-3, IL-15, TPO, EPO, SCF, Flt3L and the stimulation of gp130 and T and B-antigen receptors (Zhang et. al., 2000). Gab1 and Gab2 are also tyrosine-phosphorylated by oncoproteins such as, v-Src, Tpr-Met and Bcr-Abl (Nishida, 2003). These data suggest that the

Gab proteins act downstream of receptor tyrosine kinases and cytokine receptors. Gab2, in particular, interacts with Grb2, SHP-2, the p85 PI-3 kinase, and Shc. Grb2 has been known to constitutively interact with Gabl and Gab2 and this interaction is increased upon receptor stimulation.

Regulation of the MAPK pathway through the interaction between the protein tyrosine phosphatase SHP2 and Gab2 has been well characterized. It has been shown that mast cells and macrophages from Gab2−/− mice have decreased Erk activation in response to

SCF (Nishida et. al.,2002). Gab2–Shp2 complex also appears to have an additional, distinct signaling role in response to other stimuli. For example, over expression of

Gab2Y604F/Y633F mutant fails to bind to Shp2 blocks IL-3-evoked immediate-early gene activation without affecting Erk activation (Gu et. al., 1998).Thus Gab–SHP2 complexes act upstream of Ras, and also downstream of, or parallel to, Erk activation. SHP2 thus plays a key role in regulation of the Gab–SHP2 signaling to the Ras–Erk pathway. SHP2 contains tandem SH2 domains, the most N-terminal of which confers auto-inhibition of the C-terminal phosphatase domain (Gu, 2003; Reth et.al., 2004). Many SHP2 interaction partners including the Gab proteins contain two SHP2 binding sites, which, if phosphorylated, will act as a bi-phosphoryl tyrosine activation motif (BTAM) and confer simultaneous binding of both SH2 domains, thereby relieving auto-inhibition(Cunnick et.al.,2001). Thus the activation of the Erk/MAPK pathway occurs in response to a variety of stimuli, including treatment of cells with EGF and vascular endothelial growth factor (VEGF). Further, the Gab/SHP2 complex also positively regulates other downstream pathways. These include c-Kit-induced Rac activation, β1-integrin- and growth factor-induced PI3K activation. Research also shows the role of this complex in

VEGF-induced migration in endothelial cells as well as cell adhesion and migration of

Ba/F3 hematopoietic cells (Nishida, 2003).

Another important contribution of Gab proteins toward cellular growth and survival is their recruitment of PI-3K to activated receptors. Most, if not all, cytokines activate PI-

3K. Although many cytokine receptors have direct binding sites for the p85 subunit of PI-

3K, others, such as the interleukin-3 receptor beta common chain (betac) and the IL-2 receptor β chain (IL-2R β), lack such sites (Wöhrle et. al., 2009). It has been shown that the proto-oncoprotein Shc, also signals to the PI-3K/Akt pathway. Analysis of mutants of betac shows that the Shc binding site is required for Gab2 phosphorylation in response to cytokine stimulation. Also, when fused directly to a mutant form of IL-2R β that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Some receptors recruit PI-3K both via direct p85 binding sites and via Gab proteins, for example c-Kit and the NGF receptor. A recent study showed that a splice variant of c-Kit that recruits Gab2 induces much stronger activation of the PI-3K pathway than an isoform that does not bind Gab2 and recruits PI-3K only directly (Munugalavadla et. al.,

2007). Similarly, the B and T cell antigen receptors recruit PI-3K via Gab2. Thus, Gab proteins serve as amplifiers of PI-3K signaling in many receptor systems, in particular for those lacking direct p85 binding sites such as the IL-3 receptor.

Given their integral role in cytokine signaling, interest has also been generated to uncover their relevance in hematopoiesis. Despite the normal appearance of the mice, normal BM cellularity and the normal blood counts, Gab2-/- mice show reduced colony forming ability in methylcellulose and impaired KLS(c-Kit+Lin–Sca-1+ cell surface markers denoting mouse Hematopoietic Stem Cell) cell growth in liquid culture (Zhang et.al.,2007). These demonstrate an intrinsic requirement for Gab2 in cytokine responsiveness. The defects of these cells in response to early-acting cytokines like SCF,

TPO and IL-3 suggest that Gab2 may act as a "signal relay" protein that organizes signaling complexes and amplifies growth factor-induced receptor activation. Studies

have also shown a role for Gab2 in hematopoietic cell adhesion and migration (Yu et. al.,

2002). It was seen that enforced expression of Gab2-SHP2 binding site deletion mutant molecules dramatically reduced β1-integrin -triggered PI3 kinase activation, whereas Erk kinase activation remained unaltered. Furthermore, transduction of primary hematopoietic progenitor cells with these mutant Gab2 molecules also significantly improved their migration capacity associated with the SHP1 gene mutation.

1.4 Potential Role in Cancer

Gab proteins are implicated in a variety of human diseases. It is well-established that Gab proteins promote tumourigenesis by functioning as ‘accomplices’ of certain oncoproteins or by amplifying signaling upon their over-expression. The study of Gab1 in Met signaling and cancer has been researched in recent years. Over-expression of Gab1 promotes cell cycle progression when Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the PH domain, and the binding sites for PI-3K (Tyr-449, -474, and -591 are replaced with Phe) and SHP2, where

Tyr-659 is mutated, but not the Grb2 and CrkII/PLC γ binding sites. In this model Gab1 sufficiently promoted transformation and proliferation of fibroblasts (Mood et. al., 2006).

A role for Gab1/SHP-2 interaction in growth and transformation of NIH 3T3 fibroblasts has also been reported (Holgado-Madruga et. al, 2004).

Involvement of Gab2 in leukemogenesis was highlighted when myeloid progenitors from

Gab2-deficient mice were shown to be resistant to transformation by Bcr-Abl (Sattler et. al.,2002). Phosphorylation of Y177 within the Bcr moiety leads to recruitment of the

Grb2/Gab2 complex and downstream signaling via SHP2 and PI-3K, which is crucial for

enhanced proliferation and survival. Similarly, the oncogenic Bcr-FGFR1 fusion protein, which is also the product of a chromosomal translocation, drives the tyrosine phosphorylation of Gab2 in murine bone marrow cells and their malignant transformation through phospho-Y177 mediated Grb2 association (Roumiantsev et al., 2004). Another kinase implicated as a key component of the Bcr-Abl signaling network is Jak2 that in turn activates Lyn leading to Gab2 phosphorylation These findings highlight the role of

Gab2 phosphorylation in driving Chronic Myeloid Leukemia or CML (Scherr et. al.,

2006). After the pivotal role of Gab2 in Bcr-Abl-mediated transformation had been established, its involvement in the pathogenesis of several other leukemias was discovered. The oncogenic fusion kinases Tel-Abl and Tel-Jak2 engage Gab2 in a similar manner to Bcr-Abl(Nguyen et. al., 2001;Million et. al., 2004). Gab2 amplification has also been described in breast cancer cell lines (Daly et. al., 2002) and Gab2 mediated activation of the SHP2/Erk signaling pathway is important for the proliferation of mammary cells. Amplification of Gab2 containing region has been reported in 10-15% of human breast tumors (Bentires-Alj et. al., 2006). Various studies conducted have now demonstrated that Gab2 is not only an important interaction partner of oncoproteins involved in the transformation of hematopoietic cells, but also of those playing a well described role in solid tumours.

Given the observed role of the Gabs, especially Gab2, in cytokine signaling and coordinating interactions between signaling intermediates, I recognized an opportunity in disrupting key tyrosine-phosphorylation sites on Gab2 to further validate its interactions with its signaling partners, SHP2 and p85. Prior studies have shown how the disruption of

Gab2 binding sites for SHP2 and p85 effect cell growth, migration and activation of downstream cascades (Gu et. al.,2000;Edmead et.al.,2006; Yu et. al.,2002).

To carry out this experiment with a hematopoietic cell line, I worked with Ba/F3 cells.

These murine IL-3 dependent pro-B cells are used as a model system for assessing both the potency and downstream signaling of kinase oncogenes, and the ability of small- molecule kinase inhibitors to block kinase activity. Facilitated by their growth properties, I generated cell lines expressing retroviral constructs of wild type Gab2, along with the SHP-2 binding site mutant (Gab2Y604F/Y633F)(Gu et. al.,1998) and p85 binding sites mutant (Gab2Y441F/Y465F/Y574)(Gu et.al.,2000), using MSCV which has an Internal

Ribosomal Entry Sequence (IRES) that in turn drives the downstream GFP. These lines were biochemically used to study the binding defect that the mutants cause and also the implication of phosphotyrosine mutations on growth and survival.

2. MATERIALS AND METHODS

2.1 Purification and Sequencing of the plasmid

Plasmid DNA of MSCV-IRES-GFP-Gab2 WT, MSCV-IRES-GFP-Gab2/SHP2 mutant

and MSCV-IRES-GFP-Gab2/p85 mutant were transformed into E.Coli strain DH5α and

plated onto LB Agar plates. Single clones were picked up the next day and put for growth

O/N culture of 250 ml LB broth along with 250l of Ampicillin. The O/N cultures were

used for isolating plasmid DNA using Invitrogen’s Maxiprep kit and DNA was dissolved

in 500l of TE. DNA concentration of the clones was determined using

spectrophotometer. Primers amplifying the mutation containing regions of the Gab2

DNA were designed. Selected clones were then sent for sequencing and the returned

sequences were checked for homology with original Gab2 cDNA using NCBI BLAST.

The desired mutations were confirmed. The Hemagglutinin (HA) tag sequence was also

identified at the C-terminus of each of the clones.

2.2 Generation of producer lines and measurement of transduction efficiency

293FT cell line, which is a fast-growing, highly transfectable clonal isolate derived from

human embryonal kidney cells and is transformed with the SV40 large T antigen, was

used to carry out transient viral transfections. The 293FT cells were cultured in DMEM,

having 10% FBS and 1% antibiotic. One day before transfection, 1.5x106 cells were

seeded in a 10cm culture dish. The transfection was carried out using Invitrogen’s CaPO4

Transfection kit. 20μg insert DNA, along with 20μg pEQ-pAM3 (gag or group antigens-

pol or reverse transcriptase encoding vector) and 10μg pMDG-VSVG( that encodes for

env or viral envelope) vectors were added to the cells and left for O/N incubation at 14

37°C with 5% CO2 levels. The media on the transfected cells was changed the next day.

After 48 hours of transfection viral supernatant off the 293FT cells was collected every

12 hours, syringe- filtered using a 0.45µ filter and used to transduce ecotropic fibroblast

GP+E86 cells. The filtered supernatant containing the Gab2 and mutant retroviral constructs was added onto GP+E86 cells which were seeded at a density of 0.5x106 in

DMEM, with 10% CS and 1 % antibiotic. To this culture 1:1000 ratio of Polybrene was also added, as it is a charge negator and facilitates the entry of viral DNA into the cells.

This process was repeated over 3 days with fresh supernatant off 293FT cells to ensure that the retroviral constructs were continuously packaged and circulated into the ecotropic culture medium. After multiple rounds of transduction, the viral titer of the

GP+E86 producer cells was measured by adding different volumes of supernatant

(1mL,0.5mL,0.1mL) plus media and Polybrene to NIH3T3. The 3T3 cells were seeded at

0.1 x106 cells/ well in a 6-well dish once transduced by retrovirus, they were analyzed by

Flow Cytometry and assessed for levels of the reporter gene, GFP. A blue argon laser emitting light at 488nm was used to excite the GFP present in transduced cells. The emission spectrum was plotted in terms of Forward and Side Scatter, correlating with cell volume and complexity, respectively. These plots were used to calculate not only the amount of cells transduced by the retrovirus, but also the level of transduction, as assessed by the brightness of the emitted signal.

Flow Cytometry was also used to examine the transduction efficiency of the Gab2 WT,

Gab2/SHP2 mutant and Gab2/p85 mutant producer cell lines on WT Bone Marrow(BM) cells. BM cells from two WT female mice were harvested. The cells were then plated at a final density of 2 x 106 BM cells/ml in culture media containing IMDM with 15% FBS. 15

Recombinant cytokines IL3, IL6 and SCF were added directly to culture dish at concentration of 1 µg/µl. Gab2 retroviral producers were irradiated at 1500 Rads at the

Biomedical Research Building Irradiator Facility. 8x106 cells/ dish were seeded for transducing WT BM cells. The day after irradiating co-culture was set up between the producers and BM cells. BM cells were plated to get to the 1x106/ml mark. Media on producers was taken off and the BM cells were plated with BM culture media, cytokines and Polybrene. A portion of WT BM cells was separated as negative control. These transduced and control BM cells were then analysed for GFP levels, 48 hours following transduction.

Further, to check the level of expression of transduced Gab proteins in the producers,

Western Blot analysis was done. The producer cells were lysed using RIPA buffer (50 mM TrisHCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS ), which enabled efficient cell lysis and protein solubilization while avoiding protein degradation and interference with the proteins' immunoreactivity. The lysates were quantified and then analysed using BioRad’s SDS PAGE apparatus.

2.3 Transduction of Ba/F3

The next step was to access and validate the Gab2 mutants in a hematopoietic setting.

Although, multiple attempts were made to transduce Ba/F3 cells by either co-culturing them with retroviral producers or growing them in virus containing supernatant, this yielded no positive results. It is assumed that a blockage of the retroviral receptor could have hindered the transduction ability of these Ba/F3 cells. Another probable cause for

this behavior could be the rise and eventual dominance of a non-transducable clone that led the majority of the cells to drift towards being unresponsive to retroviral transduction.

I then switched to an alternate approach where 293FT cells were transiently transfected using PEI (from Polysciences Inc.), which is lipid based DNA delivery molecule. 1.5x106

293FT cells were seeded in a 6cm dish and a transfection mix containing either Gab2 WT or mutant retroviral constructs , packaging vector ECOPAC and 1mg/ml of PEI was added onto the cells. After 48 hours of transfection, the viral supernatant was collected and used to transduce a new batch of Ba/F3 cells ( from our collaborator R. Morrigl), which were plated at a density of 0.1x106 cells/ml in culture media containing RPMI-

1640, 10% WEHI-3-CM ( IL-3 CM) and 10% FBS. The transduced Ba/F3 cells were sorted for GFP+ cells using the BD Biosciences’ FACS Aria.

2.4 Validation of Gab2 Mutants

The expression of transduced Gab2 proteins was checked in the respective Ba/F3 cell lines. Since, all the constructs were HA tagged, it was possible to compare the over- expressed protein with its endogenous levels. Western blot analysis was done, wherein,

Ba/F3 cells expressing MSCV vector only, Gab2 WT, Gab2/SHP2 and Gab2/p85 constructs were starved of serum and IL-3 and grown in only RPMI for 5 hours. Then the cells were re-stimulated with 5ng/ml of recombinant IL-3 for 20’, after which they were centrifuged and the pellets were lysed using Triton X 100+NP40 lysis buffer (50 mM

Tris-HCl ,150 mM NaCl, 0.5% NP-40, 0.5% Triton X 100 and pH adjusted to 8.0). The lysed pellets were quantified for protein concentration and then analyzed using BioRad’s

SDS PAGE apparatus. The proteins were blotted onto PVDF membranes, which were

then probed for HA using mouse monoclonal anti-HA antibody (Sigma) and for total

Gab2 using rabbit polyclonal anti-Gab2 antibody (Millipore).

To verify how the mutations cause loss of binding of Gab2, IP + Western Blot analysis was done. The above mentioned procedure for starving and re-stimulating Ba/F3 cells was carried out. IP reactions were set-up, wherein, Protein-A-Agarose beads were used to cause the precipitation of the desired proteins. The reaction recipe included the beads, cell lysates at

1 mg/ml concentration, anti-HA antibody to ensure pull down of only transduced proteins and Triton X 100 + NP 40 lysis buffer to make up the volume to 1ml. The vials were incubated at 4° C for O/N. Following this, the beads were washed at least 3 times with the lysis buffer and then denatured and loaded onto the SDS gel for electrophoresis.

After transferring the proteins onto the PVDF membrane, the blots were then incubated and probed with respective primary antibodies, anti-Gab2, rabbit polyclonal anti-SHP2

(Millipore) and rabbit monoclonal anti-p85(Cell Signaling Tech.) followed by incubation in secondary antibodies. ECL from Pierre’s was used to develop the blots.

2.5 Dominant Negative Effect of Mutants

To assess the impact of the mutants on endogenous levels of Gab2, it was important to determine whether binding of total Gab2 was deterred due to the over-expression of tyrosine-site-mutated Gab2 constructs. I believed that the presence of the mutants would have a dominant negative impact wherein the mutated Gab2 will still be able to interact with Grb2, hence competing with the wild-type product. This competition and prevalence of mutant Gab2 would have its impact on downstream signaling.

For this, lysates of the different Ba/F3 cell lines were immuno-precipitated with anti-

Gab2 antibody, which pulled down both endogenous and transduced Gab2. The blots for the same where then probed with respective, anti-SHP2 or anti-p85 antibodies. Total amount of Gab2 was also assessed by re-probing the blots with Gab2 specific antibody.

The developed films were further analysed by densitometry, using the Image J software.

In this, respective band widths were calculated and then normalized to the area of MSCV vector only band to arrive at a relative intensity value.

2.6 Functional Impact of Mutants

The next step after biochemical verification was to study the differential pattern of growth after cytokine starvation and stimulation. Hence, the vector only GFP, Gab2 WT,

Gab2/SHP2 and Gab2/p85 cells were each divided into two batches of 2x106/10 cm dish.

Both the batches were serum and IL-3 starved for 5 hours. For each construct, one of the batches was stimulated with 1ng/ml of r-IL3 and growth patterns were observed over a timeline of 48 and 96 hours. This experiment were repeated 3 times, and also monitored along side, was the % viability of Gab2 WT cells as compared to the mutants.

3. RESULTS

Plasmid DNA for MSCV-IRES-GFP, MSCV-IRES-GFP-Gab2 WT, MSCV-IRES-GFP-

Gab2/SHP2 mutant and MSCV-IRES-GFP-Gab2/p85 mutant was purified from E.coli

DH5α clones and the presence of mutations was confirmed by sequencing. These DNA

samples along with viral packaging vectors pEQ-pAM3 and pMDG, were used to

transfect 293FT cells, by using calcium phosphate. Ecotropic GP+E86 cells were

transduced by the supernatant generated from the transient transfection of 293FT, to

create producer lines for each of the constructs. Flow analysis of the producer cells

showed that all the lines had about 98% GFP content, confirming positive transduction by

the retrovirus, and also that producer lines were capable of transducing up to 30% WT-

BM cells.

293FT cells were separately transfected with Gab2WT, Gab2/SHP2 mutant and

Gab2/p85 mutant plasmid using the lipid-based delivery vehicle, PEI. The supernatant

from these transfections was in turn used to transduce Ba/F3 cells. The Ba/F3 cells were

analysed by Flow Cytometry and it was found that the retroviral constructs had infected

about 28-30% cells. Cells positive for GFP were sorted using FACS, to generate lines

expressing MSCV-vector only, Gab2WT, Gab2/SHP2 mutant and Gab2/p85 mutant

3.1 The Gab2 Mutants Show Binding Defect toward Their Respective Partners

Our first goal was to validate the Gab2/SHP-2 binding site mutant (Gab2Y604F/Y633F) and

Gab2/p85 binding sites mutant (Gab2Y441F/Y465F/Y574) by biochemical analysis. This was

done by generating Ba/F3 cell lines for these constructs and using the cell lysates for

IP/Western Blot analysis. Once, the lines were made, we first verified the expression of

transduced and endogenous Gab2 in all of them through Western Blot (Figure 2).

Through this I was convinced that all the cell lines had comparable expression level of the desired protein and also that the antibodies were clean and pulled down the respective molecules, with minimum non-specificity. The unstimulated and stimulated cell lines were then used to carry out pull down experiments. As seen in Figure 3, when Gab2 WT and mutant cells were stimulated by IL-3, the mutants failed to bind to the respective partners. Upon cytokine stimulation and subsequent phosphorylation it was seen that

Gab2WT binds successfully to both SHP2 and p85, and both the partners were pulled down in the IP by the anti-HA antibody and detected by the respective antibodies in

Western. In contrast, Gab2/ SHP2 mutant failed to bind to SHP2 and Gab2/p85 mutant failed to bind to p85 (lanes 6 and 8, respectively). This confirmed that the retrovirally- transduced Gab2 mutant cells lack the ability to get phosphorylated at the tyrosine residues and thus cannot bind to the signaling partners.

3.2 The Presence of the Mutants has Mild Effect on the Binding of Endogenous Gab2

Through previous experiments, it has been shown that Ba/F3 cells have high endogenous levels of Gab2. I was interested to know how the transduction of these cells with constructs that deter binding, would impact the interaction of total Gab2 with its partners.

Hence, the Ba/F3 cell lines were starved and stimulated with IL-3, but this time instead of pulling down only HA tagged Gab2, total Gab2 was immuno-precipitated using anti-

Gab2 antibody. These IP samples when probed for SHP2 and p85 showed very mild binding defect. Figure 4 shows the binding ability is lower for the mutants, as compared to other lanes. The MSCV vector only cells were a useful control in this experiment as

they a clear indication of binding between only endogenous Gab2 and the partners (lane

2).

Even after almost four rounds of biochemical analysis I was unable to show a strong dominant negative effect. Thus, three sets of developed films were separately analysed through densitometry. As seen in Table 1, relative intensity of Gab2/SHP2 mutant was about 20% less than the vector only control, whereas the Gab2 WT stimulated cells were almost 99% as efficient as the control in binding and pulling down the partners. The

Gab2/p85 mutant showed about 65% lesser band intensity when compared to the control.

When this data was statistically analysed it was seen, that Gab2/p85 mutant cells showed significant defect in binding as opposed to Gab2WT cells (p value=0.03). This analysis indicated that the presence of Gab2/p85 mutant mildly effected and disrupted the interaction of endogenous Gab2 with p85.

3.3 Gab2 WT cells have No Growth Advantage Over the Mutants, but These Cells show

higher Cell Viability

The second goal of this project was to analyze stable Ba/F3 cell lines expressing wild- type Gab2 and the above mutants, for defects in cell growth and proliferation. For this experiment cell lines were starved of cytokines and serum and then re-stimulated with a very small amount of cytokine. When cell number was counted, it was observed that

Ba/F3Gab2 WT cells had about 2 fold better growth rate than the mutants (Figures 5).

Statistics revealed no significant advantage in growth by the WT cells.

As part of the same experiment, cell viability was also checked. It was seen that Ba/F3 cells expressing Gab2/SHP2 and Gab2/p85 mutants were not as viable as compared to

cells expressing Gab2WT construct ( Figure 6). Students’ T-Test showed that there was indeed a significant difference in cell viability due to the presence of Gab2-mutants (p value=0.02). This was indicative of how the disruption of Gab2 binding with its prominent signaling partners could impact cell survival.

4. DISCUSSION

Gab proteins mediate the crosstalk between various signaling pathways and can thereby

provide the basis for the synergistic action of various receptors. They function as an

interface between various receptor classes and several downstream signaling pathways

involved in the control of proliferation, cell death, migration and differentiation.

Although their major attribute is that of a positive regulator in cytokine signaling, some

in vitro studies also highlight their antagonistic properties. It has been shown that over

expression of Gab2 in T cell lines inhibits T-cell receptor evoked activation of the IL-2

promoter, blocking downstream nuclear factor of activated T-cells (NF-AT) and nuclear

factor kappa-light-chain-enhancer of activated B cells (NF-kB) directed transcription

(Pratt et. al., 2000; Parry et. al., 2005). Consequently, factors like extra-cellular ligand

availability and cell lineage play a role in governing the localization and signaling of the

Gab proteins.

Gab2 has been known to play a pivotal role in cytokine signaling. The most prominent of

its partners in hematopoiesis are predicted to be p85 and SHP2. As seen in prior

publications (Gu et. al. 1998 and 2000; Yu et. al.,2002) the binding between Gab2 and its

key partners is disrupted by mutating the Gab2 tyrosine residues into phenylalanine. Data

represented in this validates this binding defect. The significance of the phosphorylation

sites present on Gab2 not only lies in aiding its interaction with crucial binding partners,

but also on how the binding influences downstream signaling pathways like Erk, Akt, c-

Kit and c-Mpl. It is note-worthy that c-Kit and c-Mpl have been implicated in

hematological malignancies as well as HSC self renewal and quiescence. It was shown

that activation of PI-3K by c-Kit is dependent both on the direct PI3K-binding site in c-

Kit and on the phosphorylation of Gab2. The fact that c-Kit has been found mutated in numerous human malignancies, including acute myeloid leukemia, and that Gab2 is often over expressed in acute myeloid leukemia suggests a potential role of Gab2-mediated

PI3K activation in transformation (Sun et. al.,2008). TPO induced stimulation of c-Mpl has been implicated in maintaining HSC quiescence and also in myeloproliferative disorders. It has been shown how Gabs act upstream of c-Mpl and play a role in regulating PI-3K and MAPK pathways, in c-Mpl/TPO signaling (Yoshihara et al., 2007;

Bouscary et. al., 2001).

It was seen in this study that over-expression of the mutants shows a very distinct binding defect when the transduced proteins are immunoprecipitated along with HA-tagged

Gab2, but this defect mildly interfered with endogenous Gab2 binding to its partners, especially p85. Since the transduced Ba/F3 cell lines used for this study constitutively express the mutants and I suspect that the defect in binding is likely to get over-shadowed by the presence of the endogenous Gab2. The Gab2/SHP2 and Gab2/p85 mutant- expressing cell lines have considerable expression of un-mutated endogenous Gab2 that compensates for the loss of binding, thus making the defect less apparent. A similar pattern is seen when growth response is measured after starvation and cytokine re- stimulation. Although, the mutants confer less survival and cause the cells to grow at a slower rate, the fold difference between their growth rates and the WT cells is less than 2 fold. But cell viability, on the other hand, is significantly affected by the presence of the

Gab2-mutants. Data shows a presence of higher number of viable Ba/F3 Gab2WT cells as compared to the mutants (Figure 6). This indicates how the inability of Gab2 to bind to

its signaling partners has an effect on cell survival, more so due to the role of pathways like PI-3K in regulating cell death and viability.

Since, the Gab2/SHP2 and Gab2/p85 mutants have now been biochemically assessed as a part of this study, I feel that these mutants can be further used to dissect the structure- function relationship of Gab2 in vivo in KO models, where there will be no background endogenous Gab2. These add back experiments will clarify the physiological significance of Gab2 phosphorylation and its subsequent impact on downstream molecules like

STAT5 and Akt, in a clean Gab2-/- setting.

Gab2 is highly associated with hematopoietic growth factor receptors and its deficiency is likely to play a role in aberrant cytokine stimulation of hematopoietic cells. Previous work in our lab has also indicated that Gab2 deficiency leads to significantly reduced multilineage hematopoietic repopulating activity (Zhang et. al., 2007). As part of future studies, a retroviral complementation approach using the tyrosine-binding site mutants will help in investigating the role Gab2 interaction with p85 and SHP2 in hematopoiesis.

This will help in determining whether Gab2 requires p85 and SHP2 binding sites in order to effectively promote hematopoietic reconstitution in a transplant setting. I expect that loss of these interactions will ablate Gab2 function below that obtained with wild-type

Gab2 and also ameliorate the effects of Gab2 on cancer signaling pathways. However, it is possible that the repopulating activity of mutant Gab2 lacking these sites will still be better than complete deficiency due to the known positive role for SHP-2 in hematopoiesis and possibly due to its ability to promote Erk activation. Because of the big defects associated with Gab2 deficiency, comparison of each mutant with the other is also possible. Another possibility can be to consider developing double p85 and SHP-2

mutant Gab2 vectors for analysis to determine if both binding sites are required for full activity.

Potential mechanisms that may strengthen the action of phosphorylation-dependent positive or negative feedback on Gab proteins have been characterized. Firstly, phosphorylation of a particular residue might affect the phosphorylation of a nearby residue in either a positive or antagonistic fashion, due to phosphorylation-induced changes in protein conformation. Secondly, phosphorylation-induced conformational changes may alter the accessibility of key regions, such as the PH domain.

That is why in addition to the mutants discussed in this study, I have also generated

Gab2S160A (Akt) and Gab2S623A (Erk) point mutants. Studies show negative feedback regulation of Gab2 by serine phosphorylation at sites near the PH domain (Akt) or near the SHP-2 binding domain (Erk). Akt has been shown to constitutively associate with

Gab2, phosphorylate it on a consensus phosphorylation site (Ser159) and inhibit Gab2 tyrosine phosphorylation. Mutation of S159A (corresponding mutation in mouse is S160) resulted in increased tyrosine phosphorylation of Gab2 and the Gab2S159A mutant displayed transforming properties in fibroblasts and prolonged signaling through the PI-

3K/Akt pathway. This might impact downstream STAT5 activity, as it has been shown that constitutively active STAT5 forms a complex with the p85 subunit of the PI3-K and

Gab2 in leukemic bone marrow cells, resulting in the activation of Akt (Harir et. al.,

2007). Gab2 is also regulated by ERK-mediated negative feedback phosphorylation, wherein the identified S623 is the site of phosphorylation of Gab2 by ERK. Studies have shown that the Gab2/SHP2 interaction is enhanced by S623A mutation. This is in turn expected to strongly inhibit downstream STAT5. Thus, it will be of relevance to study the

balance between positive and negative signaling through both in vitro models and further complement it by in vivo analysis. Negative feedback is deemed critical in shifting Gab2 signaling from Erk and Akt to STAT5. This mechanism may be particularly relevant during conditions of hematopoietic stress such as recovery from myelo-suppression or leukemic hematopoiesis. These conditions are more likely to increase Gab2-mediated

ERK and Akt signaling, while inhibiting STAT5, whereas blockage or complete deficiency of Gab2 are expected to have a reverse effect.

Since Gabs bind many common receptors and are very under studied in hematopoiesis, it is pivotal to understand the role of binding to their partners and how it impacts oncogenic tyrosine kinase signaling. As cytokine signaling pathways are dysregulated in hematologic disease, a better understanding of the regulation of signaling intensity and duration are keys to uncovering new drug therapies.

FIGURES AND TABLES

Figure 1: Gab2 interactions with binding partners Diagram shows generic Gab adapter docked at the plasma membrane by the PH domain. Interactions with p85 and SHP-2 are involved in Akt and Erk activation respectively. Binding of Receptor Tyrosine Kinases to their receptive ligands the kinase activity of the cytoplasmic domain of the receptor. The receptor becomes phosphorylated on tyrosine residues. Docking proteins such as Grb2 contain SH2 domains that bind to the phosphotyrosine residues of the activated receptor. Grb2-Gab interact through the SH3 domains and activate downstream signaling pathways, PI-3K/Akt and SHP2/Erk. Negative feedback by serine phosphorylation of Gab at S160 by Akt and S623 by Erk (block arrows) plays an important role in control of function and signaling of Gabs.

- + - + - + - + IL-3 GFP Gab2 WT G2/SHP2 G2/p85 1 2 3 4 5 6 7 8

Figure 2: Uniform Gab2 expression levels seen in Ba/F3 cells expressing WT, G2/SHP-2 binding site mutant (Gab2Y604F/Y633F) and G2/p85 binding site mutant (Gab2Y441F/Y465F/Y574) MSCV-Vector only, Gab2, G2/SHP2 mutant and G2/p85 mutant transduced Ba/F3 were serum starved for 5 hr and stimulated for 20’ with 5 ng/ml of r-IL3. Cells lysates were prepared in Triton+NP40 buffer and samples were immunoblotted for HA tagged Gab2 and total Gab2, using anti-HA and anti-Gab2 antibodies, respectively. Anti-actin antibody was used to verify the Western Blot loading control.

- + - + - + - + IL-3 GFP Gab2 WT G2/SHP2 G2/p85 1 2 3 4 5 6 7 8 9

Figure 3: Transduction of Ba/F3 cells with mutated Gab2 constructs results in defective binding of Gab2 with SHP2 and p85 Ba/F3 cells expressing vector-only, Gab2, G2/SHP2 and G2/p85 retroviral constructs were serum starved for 5 hr and stimulated for 20’ with 5 ng/ml of r-IL3. Cell lysates were prepared and anti- HA immunoprecipitation(IP) was performed. Immunoprecipitates were resolved by SDS-PAGE and then immunoblotted(IB) with anti-HA, anti-p85 and anti-SHP2 antibodies. Ba/F3 parental cell lysates was used as positive control for Western blotting.

- + - + - + - + IL-3 GFP Gab2 WT G2/SHP2 G2/p85

1 2 3 4 5 6 7 8 9

Figure 4: Expression of G2/SHP-2 binding site mutant (Gab2Y604F/Y633F) and G2/p85 binding site mutant (Gab2Y441F/Y465F/Y574) mutants show mild defect on total Gab2 binding with the partners, SHP2 and p85 Ba/F3 cells transduced with Gab2, vector-only, G2/SHP2 or G2/p85 mutants were serum starved for 5 hr and stimulated for 20’ with 5 ng/ml of r-IL3. Triton X100+NP-40 buffer was used to prepare cell lysates. Anti-Gab2 antibody was used to perform immunoprecipitation (IP) of these experiments, to pull down both endogenous and retrovirally expressed Gab2. The samples were immunoblotted with anti-p85 and anti-SHP2 antibodies. The blots were stripped and re-probed with anti-Gab2. Developed films were further analysed by densitometry, which revealed that Gab2/p85mutant mildly affects the binding of endogenous Gab2 with p85.

Growth Rate (Fold Change) (Fold Rate Growth

Figure 5: Ba/F3 Gab2WT cells show no growth advantage over Gab2/SHP2 and G2/p85 mutant samples, after cytokine withdrawl and subsequent IL3 stimulation Ba/F3 cells expressing only IR-GFP, Gab2 WT and the two tyrosine point mutants Gab2/SHP2 and Gab2/p85 were serum starved for 5 hours and stimulated with 1ng/ml of r-IL3. Cell numbers were counted using a hemocytometer and fold change in growth rate was monitored at 48-96 hour time-points. Statistical analysis revealed no significant difference in growth patterns when Gab2WT samples are compared with mutant samples, using student’s T-Test.

viable cells (in %) (in cells viable

- Non

Figure 6: Ba/F3 Gab2 cells show significantly higher cell viability compared to mutant samples, after IL3 stimulation Ba/F3 cells expressing only IR-GFP, Gab2 WT or the two tyrosine point mutants Gab2/SHP2 and Gab2/p85 were serum starved for 5 hours and stimulated with 1ng/ml of r-IL3. After cytokine stimulation the % Non-viable BaF3 cells were determined by light microscopy using a hematocytometer and Trypan blue staining, at the 48hour and 96hour time-points. Gab2WT sample data was statistically compared with Gab2/p85 and Gab2/SHP2 data, using student’s T- Test.

Table 1: Densitometric analysis of Gab2, G2/SHP2 and G2/p85 reveals a mild defect in interaction of total Gab2 with its respective partner when over-expressing G2/p85 mutant, but not G2/SHP2 mutant Ba/F3 cells expressing Gab2 WT and G2/SHP2 and G2/p85mutant constructs were starved for 5 hours and stimulated with 1ng/ml of rIL-3. These cell lysates were immunoprecipitated using anti-Gab2 antibody and immunoblotted using respective antibodies for SHP2 and p85, as seen in Figure 4. Densitometric analysis of the developed films was done using Image J software. This data is average of three independent experiments.

Area of Peak GFP stim. Gab2 stim. G2/SHP2 stim. G2/p85 stim.

Gab2-SHP2 15897.99 16911.37 12981.07 15325.31 Total Gab2 7659.98 26330.39 24810.23 24526.12 Ratio ( SHP2:Total) 2.08 0.64 0.52 0.62 Relative intensity 1 1.06 0.82 0.96 Std. Dev ( SHP2: Total) 0.74 pvalue (Gab2 v/s G 2/SHP2) 0.27

Area of Peak GFP stim. Gab2 stim. G2/SHP2 stim. G2/p85 stim.

Gab2-p85 7427.80 9854.65 9607.43 2995.53 Total Gab2 3998.29 28992.08 28707.74 21415.15 Ratio ( p85:Total) 1.86 0.34 0.33 0.14 Relative intensity 1 1.33 1.29 0.40 Std. Dev ( p85: Total) 0.80 pvalue ( Gab2 v/s G2/p85) 0.03

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