The role of paxillin superfamily members hic 5 and leupaxin in b cell antigen receptor signaling 1

ABBREVIATIONS ARF ADP-ribosylation factor BCR B cell receptor BLNK B cell linker protein Btk Bruton’s tyrosine kinase Csk C-terminal Src tyrosine kinase DAG Diacylglycerol DNA Deoxyribo

THE ROLE OF PAXILLIN SUPERFAMILY MEMBERS- HIC-5 AND LEUPAXIN

IN B CELL ANTIGEN RECEPTOR

SIGNALING

CHEW SUK PENG

NATIONAL UNIVERSITY OF SINGAPORE

2007

THE ROLE OF PAXILLIN SUPERFAMILY MEMBERS- HIC-5 AND LEUPAXIN

IN B CELL ANTIGEN RCEPTOR

SIGNALING

CHEW SUK PENG

BSc PHARMACY (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

NUS GRADUATE SCHOOL FOR INTEGRATIVE

SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE

ACKNOWLEDGEMENTS

I would like to express my heartfelt appreciation to my supervisor Associate Prof Lam Kong Peng for his guidance and critical comments throughout the entire project I’m grateful to my fellow colleagues especially, Ng Chee Hoe, Andy Tan Hee Meng and Dr Joy Tan En Lin for their technical assistance I’m also thankful to other members of the lab including Dr Wong Siew Cheng, Lee Koon Guan, Dr Yap An Teck, Dr Hou Jian Xin and Dr Xu Sheng Li for their constant insightful comments and suggestions to my project Special thanks to attachement students Lin You Bin, Xianne Leong, Lionel Low and Sharon Goh for their friendship and encouragement Appreciation is also extended to lab biologists Chew Weng Keong, Tan Kar Wai, Chan Siow Teng and Elaine Tan for their contribution in managing the lab and allowing smooth progress of the project

To my family members my mom, my aunt, my uncle and cousins thanks for their encouragement, moral supports, love and concerns especially for taking good care of me and tolerating my busy schedule and occasional bad temper and mood swing My fellow PhD mates from A*Star Graduate Scholarship, especially Pauline Tay, Liu Mei Hui, Tam Wai Leong, Dave Aw, Cecilia Lee, Lee Terk Shuen, Harmeet Singh, Adrian Mathew Mak, Emril Mohamad Ali, Fong Siew Wan and Sebastian Ku,

I truly cherish their constant support and occasional social meetings to complain and listen to each other about difficulties and stress in research My personal friends, Franck M, Harry Chua, Angel Choong, Kristie Ong, Eryn Chew, Angela Koo, Jessey Ding, Lynda Lee, Chin Woey, Jacqueline Chong, Jerry Tan, Lim Thian Yew, Dave

Chia and Simon Heng, thanks for their constant support and having the faith in me to complete my PhD Finally, special thanks to a special friend, Jackson Chiam, for his love and support

I thank God for without His grace and blessing I would not have come this far Also thanks to my church friends especially Grace, Cecilia, Sabrina, Victor and Carmen for their constant prayers

1.6 Negative regulatory pathways of B cell antigen receptor signaling 26

CHAPTER 2: MATERIAL AND METHODS

2.1 List of antibodies for fluorescence, BCR stimulation,

2.3.13 Transformation of DH5α by heat shock method 58 2.3.14 Bacterial DNA mini-prep by alkaline lysis 58 2.3.15 Bacterial maxi-prep using Qiagen Maxi-prep columns 59

2.5 Molecular and cellular immunology methodology 62

2.5.3 BCR-induced activation of IL-2 promoter 63

CHAPTER 3: THE ROLE OF HIC-5 IN B CELL RECEPTOR SIGNALING

3.2.1 Yeast-two-Hybrid using B cells adaptor protein, Bam32 as a bait 72

3.2.3 Interaction of Bam32 with Hic-5 and its homologue, paxillin 74

3.2.4 Interaction of Bam32 homologues: TAPP1 and TAPP2, with Hic-5

3.2.5 PH domain of Bam32 mediates binding to Hic-5 and paxillin 78 3.2.6 Interaction of Hic-5 and paxillin with Lyn is independent of Bam32

81 3.2.7 Bam32 competes with Hic-5 and paxillin to interact with Lyn 82 3.2.8 Tyrosine phosphorylation of Hic-5 and paxillin by Lyn in HEK293T

3.2.9 BCR-induced tyrosine phosphorylation of Hic-5 87 3.2.10 BCR-induced interaction of Hic-5 with Lyn 90 3.2.11 Hic-5 was recruited to the plasma membrane upon BCR ligation 91 3.2.12 Inhibition of JNK and p38 activation by Hic-5 in A20 B cells 94

4.2.3 Leupaxin is recruited to the plasma membrane upon BCR ligation in

4.2.5 Leupaxin interacts with Lyn through its LD3 domain 117 4.2.6 Lyn phosphorylates leupaxin at tyrosine 72 119 4.2.7 Selective inhibition of JNK, p38 and Akt pathways by leupaxin in A20

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SUMMARY

Adaptor proteins play an important role in B cell antigen receptor (BCR) signaling by mediating intermolecular interactions in a spatial and temporal manner One of these adaptor proteins, Bam32, has been shown to regulate BCR signaling On the other hand, the role of paxillin superfamily of adaptor proteins in BCR signaling has not been studied previously Paxillin superfamily members consist of paxillin, Hic-5 and leupaxin based on their homology in multiple amino (N)-terminal leucine (L)- and aspartate (D)-rich sequences (LD domains) and carboxyl (C)-terminal lin-11, isl-1, mec-3 (LIM) domains Both LD and LIM domains allow protein-protein interactions The role of paxillin superfamily adaptor proteins, in particular paxillin and Hic-5, is well established in growth factor and integrin mediated signaling pathways In this thesis, the potential role of paxillin superfamily members - Hic-5 and leupaxin in BCR signaling were explored

The project was initiated by a yeast-two-hybrid screen using Bam32 as a bait, which identified Hic-5 and Lyn as potential binding partners Later we found that Hic-5 can also interact with Lyn, which is a critical Src-family kinase in BCR signaling Our current discoveries lead us to a model where Hic-5 is recruited to the plasma membrane and binds Lyn upon BCR signaling Following that Hic-5 is tyrosine phosphorylated and hence activated by Lyn By overexpression in mouse A20 lymphoma B cells, we showed that Hic-5 is a negative regulator in BCR signaling specifically in the phosphorylation of JNK and p38 MAPK Bam32 by competing with Hic-5 to bind Lyn regulates the inhibitory function of Hic-5

in the interaction with Lyn Of a total of 11 tyrosine (Y) sites on LPXN, we mutated Y22, Y72, Y198 and Y257 to phenylalanine (F) and demonstrated that LPXN was phosphorylated by Lyn only at Y72 and this tyrosine site was proximal to the LD3 domain of LPXN, which is the domain responsible for its interaction with Lyn The overexpression of LPXN in A20 B cells led to the suppression of BCR-induced activation of JNK, p38 MAPK and to a lesser extent, Akt but not Erk and NFkB, suggesting that LPXN could selectively repress BCR signaling We further showed that LPXN suppressed the secretion of IL-2 by BCR-activated A20 B cells and this inhibition was abrogated in the Y72F LPXN mutant, indicating that the phosphorylation of Y72 is critical for the biological function of LPXN in B cells

In conclusion, we discovered a previously unknown inhibitory function of paxillin superfamily adaptor proteins in BCR signaling

ABBREVIATIONS

ARF ADP-ribosylation factor

BCR B cell receptor

BLNK B cell linker protein

Btk Bruton’s tyrosine kinase

Csk C-terminal Src tyrosine kinase

DAG Diacylglycerol

DNA Deoxyribonucleic acid

Dok Downstream of tyrosine kinases

ERK Extracellular-signal-regulated kinase

FACS Florescence activated cell sorting

FAK Focal adhesion kinase

FITC Fluorescein isothiocyanate

IRS Insulin receptor substrate

ITAM Immunoreceptor tyrosine-based activation motif

ITIM Immunoreceptor tyrosine-based inhibitory motif

JNK c-Jun N-terminal kinase

LD Leucine (L) and aspartate (D)-rich

LIM lin-11 (L), isl-1 (I) and mec-3 (M)

Lyn Lck/yes-related novel tyrosine kinase

MAPK Mitogen activated protein (MAP) kinase

MHC Major histocompatibility complex

NFAT Nuclear factor of activated T-cells

NF-κB Nuclear factor κB

PEP PEST domain tyrosine phosphatase

PI(3,4)P2 Phosphatidylinositol 3,4-bisphosphate

PI(3,4,5)P3 Phosphatidylinositol 3,4,5-triphosphate

PI3-K Phosphatidylinositol 3-kinase

PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate

PCR Polymerase chain reaction

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PTEN Phosphatase and tensin homolog deleted on chromosome 10 PTK Protein tyrosine kinase

PTP Protein tyrosine phosphatase

Pyk2 Proline-rich tyrosine kinase 2

RasGAP Ras GTPase activating protein

RasGEF Ras-guanine nucleotide exchange factor

RasGRP Ras-guanine nucleotide releasing protein

SFKs Src family kinases

SH2 Src-homology 2

SH3 Src-homology 3

Shc SH2 domain -containing transforming protein C

SHIP-1 SH2-containing inositol 5’-phosphatase-1

SHP-1 SH2-domain containing protein tyrosine phosphatase-1

SOS Son od sevenless protein

Syk Spleen-associated tyrosine kinase

TAPP Tandem PH domain-containing protein

TCR T cell receptor

TLR Toll-like receptor

TNF- Tumour nerosis factor alpha

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LIST OF SCHEMATIC DIAGRAMS AND TABLES

Diagram 1.1 General overview of BCR signaling pathways

Diagram 1.2 Structural features of Src family kinases

Diagram 1.3 Schematic illustration of activation and inactivation of Src

kinase via phosphorylation and dephosphorylation Diagram 1.4 A list of adaptor proteins identified in lymphocytes

Diagram 1.5 Schematic illustration of the major structural features of

Bam32 Diagram 1.6 Model of Bam32 functions in B cell activation

Diagram 1.7 Schematic illustration of homology between Bam32, TAPP1

and TAPP2 Diagram 1.8 Positive and negative roles of Lyn in BCR signaling

Diagram 1.9 Schematic illustrations of the structural figures among

three paxillin superfamily members Diagram 1.10 The role of paxillin superfamily adaptors proteins

downstream of growth factors and integrins signaling pathway

Table 4.1 Prediction result for potential tyrosine phosphorylation

sites in LPXN using NetPhos 2.0 server

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LIST OF FIGURES

Figure 3.1 Interaction of Bam32 with Lyn

Figure 3.2 Interaction of Bam32 with Hic-5, a paxillin superfamily member Figure 3.3 Interaction of Bam32 with Hic-5 and paxillin, members of paxillin

superfamily Figure 3.4 Homology between Bam32, TAPP1 and TAPP2

Figure 3.5 Interaction of Bam32 homologues: TAPP1 and TAPP2, with Hic-5 Figure 3.6 Interaction of Bam32 homologues: TAPP1 and TAPP2, with

paxillin Figure 3.7 Generation of Bam32 truncation mutants

Figure 3.8 PH domain of Bam32 mediates binding to Hic-5

Figure 3.9 PH domain of Bam32 mediates binding to paxillin

Figure 3.10 Hic-5 and paxillin can interact with Lyn independent of Bam32 Figure 3.11 Bam32 competes with Hic-5 and paxillin to bind Lyn

Figure 3.12 Phosphorylation of Hic-5 and paxillin by Lyn in HEK293T

Figure 3.13 Competitive nature of Bam32 with Hic-5 or paxillin for

phosphorylation by Lyn Figure 3.14 Phosphorylation of Hic-5 upon BCR ligation

Figure 3.15 No tyrosine phosphorylation of paxillin detected upon BCR

ligation Fig.ure 3.16 Phosphorylation of FLAG-tagged Hic-5 in A20 upon BCR ligation Figure 3.17 BCR induced binding of Hic-5 with Lyn

Figure 3.18 Hic-5 is recruited to the plasma membrane upon BCR ligation Figure 3.19 Expression of various proteins in A20 B cells transfected with

various plasmids

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Figure 3.20 Effect of Hic-5, Bam32 or both Hic-5 and Bam32 over-expression

in MAPKs activation in A20 cells Figure 4.1 Sequence consensus between human and mouse LPXN

Figure 4.2 Expression of LPXN in several human B cell lines

Figure 4.3 Tyrosine phosphorylation of Leupaxin in BJAB B cells

Figure 4.4 Tyrosine phosphorylation profile of LPXN as compared to that of

Lyn upon BCR ligation Figure 4.5 Membrane recruitment of Leupaxin upon BCR ligation

Figure 4.6 Interaction of LPXN with Lyn in HEK293T

Figure 4.7 Interaction of LPXN with Lyn upon BCR ligation in BJAB cells Figure 4.8 Colocalization of LPXN with Lyn upon BCR ligation

Figure 4.9 A schematic illustration of plasmids constructs with truncations or

specific deletion of Leupaxin LDs domains Figure 4.10 LPXN interacts with Lyn via its LD3 domain

Figure 4.11 Tyrosine phopshorylation of paxillin superfamily members by Lyn

in HEK293T cells Figure 4.12 Schematic illustration of the positions of 11 tyrosine residues in

Leupaxin Figure 4.13 Tyrosine site 72 is important for tyrosine phosphorylation of

LPXN by Lyn Figure 4.14 The Y72F mutant of LPXN can still bind Lyn

Figure 4.15 Level of extopically expressed LPXN versus the endogenous

protein in A20 B cells Figure 4.16 Leupaxin inhibits phosphorylation of JNK and p38 MAPK but not

Erk upon BCR ligation Figure 4.17 Leupaxin inhibits phosphorylation of Akt upon BCR ligation Figure 4.18 Lack of effect of LPXN on NF-κκκκB activation

Figure 4.19 Expression of different amounts of LPXN in A20 B cells

Figure 4.20 Dosage effect of Leupaxin in suppression of BCR-induced IL-2

production in transfected A20 B cells Figure 4.21 Suppression of BCR-induced IL-2 promoter activation by A20 B

cells overexpressing LPXN

Figure 4.22 Y72 is important for LPXN phosphorylation in A20 cells

Figure 4.23 Overexpression of various FLAG-tagged LPXN plasmids in A20

cells Figure 4.24 Y72 is important for the inhibitory role of LPXN in BCR-induced

IL-2 production Figure 4.25 Y72 is important for the role of LPXN in suppressing BCR-

induced IL-2 promoter activation

ix

LIST OF PUBLICATIONS

Valerie Chew and Kong-Peng Lam

Leupaxin negatively regulates B cell receptor signaling

J Biol Chem 2007 Sep 14;282(37):27181-91 Epub 2007 Jul 19

Valerie Chew, Xiao Xing Cheng and Kong-Peng Lam

Hic-5, a Bam32 and Lyn interacting protein which plays a negative regulatory role in

B cell receptor signaling

(Manuscript in preparation)

Valerie Chew, Xiao Xing Cheng and Kong-Peng Lam

Hic-5, a Bam32 interacting protein and a negative regulator in BCR signaling

(Abstract for poster published in 16 th European Congress of Immunology, Paris, 2006)

CHAPTER 1 INTRODUCTION

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1.1 The immune system

The immune system is an interactive network of lymphoid organs, immune cells, humoral factors and cytokines which protects us from daily exposure to invading organisms It identifies and kills pathogens ranging from viruses, bacteria and parasites as well as tumor cells which are identified as foreign Immunity can be divided into two major parts - the innate and adaptive immunity, depending on their 1) specificity, 2) memory and 3) speed of the reaction The immune system is to be kept

in a well balanced manner to protect over-reactivity which causes autoimmune or hypersensitivity diseases as well as to avoid under-reactivity which causes immunodeficiency (Parkin and Cohen, 2001)

1.2 Innate and adaptive immunity

The innate immunity is the first line of defence in immune response Although

it is immediate, it is rather non specific and has no memory after the response The innate response involves several mechanisms including physical, chemical and microbiological barriers as well as immune components such as neutrophils, monocytes, macrophages, complements, cytokines and acute phase proteins (Dempsey et al., 2003) For instance, our skin is a good physical barrier while tears and saliva make a good chemical barrier against various invading organisms Immune cells such as neutrophils and macrophages are able to kill invading microbes by phatocytosis Complements on the other hand, are able to kill microbes via

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opsonization and complement-mediated lysis Due to its non-antigen specific nature, innate immunity may cause damages to normal tissues especially when non-specific inflammation is triggered with various cytokines or complements Despite their non-specificity, the innate immune system does provide some degree of discrimation between foreign molecules from self Phagocytes are able to recognize certain pathogen-associated molecular patterns on invading microbes such as lipopolysaccharide on gram positive bacteria, lipotechoic acid on gram negative bacteria, and mannens on yeast cell walls The cells involved in innate immunity carry three types of pattern-recognition receptors according to their functions: first, those enhancing endocytosis and antigen-presentation; second, those activating nuclear factor κB (NFκB) and promoting cell activation (toll-like receptors) (Muzio

and Mantovani, 2001) and third, those enhancing opsonization

In contrast to innate immunity, adaptive immunity comprises a more sophiscated immune reaction against invading microbes Adaptive immunity is a hallmark of higher order animals and comprises antigen-specific reactions However,

it is slower to respond as compared to innate immunity and it usually takes several days to weeks to develop Unlike the innate immunitry, adaptive immunity has memory which makes subsequent exposure to the same antigen faster and more vigorous (Parkin and Cohen, 2001) The major players in adaptive immunity are T and B lymphocytes or also known as T and B cells Briefly, targeted effector responses are triggered after the antigen is presented to and recognized by the antigen-specific receptors on T and B cells T and B cells upon activation will undergo cell priming, activation, proliferation and differentiation within the

3

specialized environment of lymphoid tissue Soon after, T cells are able to leave the lymphoid tissues and travel systemically to site of infection and exert their effector responses Two major types of T cells are CD4+ T cells (also known as T helper cells) which can be subdivided to CD4+ Th1 cells that help in macrophages activation and CD4+ Th2 cells that help in B cells activation; whereas CD8+ T cells (also known as

T cytotoxic cells) are known to be able to kill infected cells directly (Parkin and Cohen, 2001) Meanwhile activated B cells could proliferate and differentiate into plasma cells which secrete antibody specific to the antigen, hence leading to the eradication of infectious agents Various actions of antibody include neutralizing toxins, activating complements, enhancing opsonization of bacteria for phagocytosis and sensitizing infected or tumour cells for antibody-mediated cytotoxicity by killer cells (Parkin and Cohen, 2001)

Our current project focuses on B cell receptor signaling pathways with the identification of a novel family of negative regulators in B cell receptor (BCR) signaling Ligation of BCR with antigen triggers a cascade of BCR signaling pathways which are important for B cells activation, proliferation and differentiation Therefore more details regarding BCR signaling pathways will be discussed in subsequent sections

1.3 B cell antigen receptor signaling pathways

B cell receptor (BCR) ligation with antigen triggers a cascade of signaling events involving the activation of three major protein tyrosine kinases (PTKs) namely

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the Src family, Syk family and Tec family kinases (Kurosaki, 1997) Lipid raft which

is also known as glycolipid-enriched microdomains (GEM) in the plasma membrane, play an important role in BCR signaling (Cheng et al., 2001) BCR ligation results in translocation of the receptor to raft where key components of BCR signaling such as Src kinase, Lck/yes-related novel tyrosine kinase (Lyn) reside The result of this concentration of B cell receptor and effector proteins in raft which specifically includes and excludes different proteins allows BCR signaling to propagate within the defined fraction on the B cell membrane (Cherukuri et al., 2001; Simons and Toomre, 2000)

Besides translocation of BCR to raft, one of the earliest events of BCR signaling involves the activation of Src-family kinases such as Lyn (Burkhardt et al., 1991; Wechsler and Monroe, 1995; Yamamoto et al., 1993; Yamanashi et al., 1992) Following that, Src-family kinases are known to phosphorylate immunoreceptor tyrosine-based activation motif (ITAM) within the cytoplasmic domains of immunoglobulin (Ig)- and Ig- , which are the Ig complexed to BCR (DeFranco et al., 1995) (Details illustrated in section 1.5) The ITAM consists of YxxL/I-x-YxxL/I (where Y is tyrosine, L is leucine, I is isoleucine and x is any residue) (Colonna et al., 2000; Daeron, 1997; Moretta et al., 2001) The phosphorylated ITAM then recruites and activates Spleen-associated tyrosine kinase (Syk) (Burg et al., 1994; Hutchcroft et al., 1992) an essential protein that transduces BCR stimulation to downstream activation of various proteins like B cell linker protein (BLNK), Phospholipase C gamma 2 (PLCγ2), Phosphatidylinositol 3-kinase (PI3K) and the Tec family kinase,

Bruton’s tyrosine kinase (Btk) (Dal Porto et al., 2004) The results of that is the

5

regulation of several transcription factors that govern gene transcriptions in B cells in response to BCR ligation (Tsubata and Wienands, 2001) Such responses include B cells activation, proliferation and differentiation that help in the eradication of infectious agents

BCR signaling pathways are further modulated by several other coreceptors

on the membrane which include CD19 (positive regulator) as well as CD22 and

FCγRIIB1 (negative regulators) (Dal Porto et al., 2004) CD19 plays a role in

recruiting and hence activating Lyn, PI3K and Btk which enhance the BCR signaling pathways CD22 and FCγRIIB1 negatively regulate BCR signaling by recruiting

several phosphotases that help to dephosphorylate and hence deactivate proteins activated upon BCR ligation (Veillette et al., 2002) As reviewed in Diagram 1.1 below, BCR signaling activates four major downstream pathways: PI3-K, PLCγ2, Ras/Raf/Erk, and Vav/Rac These pathways will be discussed in more details in the next section The negative regulation of BCR signaling will also be discussed further

in section 1.7:

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Diagram 1.1: General overview of BCR signaling pathways BCR ligation triggers

first the activation of Src family kinase particularly Lyn which then leads to activation of Syk, BLNK, Btk and PLCγ2 These proteins trigger downstream

generation of second messengers like IP3 and Ca2+ which in return activate various gene transcriptions in response to the various signals received Some co-receptors involved in regulating BCR signaling include CD19, CD22, FCγRIIB1 and CD45

(indicated in yellow) The complexity of BCR signaling pathways allows dynamic B cells functions such as proliferation, survival, differentiation and apoptosis (Diagram obtained from Cell Signaling Techonology website)

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1.3.1 Signaling via PI3-K pathway

PI3K is an important kinase activated upon BCR ligation BCR co-receptor molecule CD19 was thought to play a major role in recruiting PI3K to lipid raft and hence its activation upon BCR ligation (Gold et al., 2000; Wang et al., 2002) The mechanism involves Lyn phosphorylation of the cytoplasmic tail of CD19 which subsequently recruits Src-homology 2 (SH2) domain of p85 adaptor subunit of PI3K (Buhl and Cambier, 1999; Tuveson et al., 1993; Fujimoto et al., 1998) Upon activation, PI3K phosphorylates the membrane lipid Phosphatidylinositol 4,5-biphosphate [PI(4,5)P2)] to Phosphatidylinositol 3,4,5-triphophate [PI(3,4,5)P3] (Cantrell, 2002) PI(3,4,5)P3 is essential for recruitment of several Pleckstrin homology (PH)-domain containing downstream signaling proteins such as Btk (a Tec family kinase), protein kinase B (PKB) (also known as Akt), PLCγ2 and Bam32 (a B

cell adaptor protein) to lipid raft (Niiro and Clark, 2002)

A point mutation in the PH domain of Btk leads to defects in its recruitment to PI(3,4,5)P3 and hence defects in BCR signaling and B cell maturation- resulting in a condition termed X-linked immunodeficiency (Xid) in mice (Cancro et al., 2001; Rawlings et al., 1993; Takata and Kurosaki, 1996) A similar condition in human termed X-linked aggamaglobukinaemia (XLA) is also associated with multiple Btk mutations (Tsukada and Witte, 1994; Vorechovsky et al., 1995) Btk activation will lead to downstream activation of PLCγ2 which will be addressed in greater details in

the next section

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Activation of Akt by PI3K is well established and it involves the binding of N-terminal PH domain of Akt to PI(3,4,5)P3 generated by PI3K upon BCR ligation (Andjelkovic et al., 1997; Astoul et al., 1999; Bellacosa et al., 1998) The recruitment

of Akt to lipid raft results in conformational changes that facilitate phosphorylation of its threonine 308 and serine 473 residues that are critical for full Akt activation (Bellacosa et al., 1998; Cantrell, 2002) Substrates of Akt include Bcl-2-associated death promoter (Bad), Glycogen synthase kinase 3 (GSK-3) and it also plays a role in regulating Nuclear factor κB (NF-κB) activity (Cantrell, 2002; Dal Porto et al., 2004)

Brieftly, Akt was thought to play an important anti-apoptotic role by phosphorylating Bad, a pro-apoptotic protein (Datta et al., 1997; del Peso et al., 1997) Upon phosphorylation by Akt, Bad dissociates from Bcl-XL (anti-apoptotic molecule) and binds to 14-3-3 This releases Bcl-XL and results in cell survival Akt also inhibits GSK-3, a multifunctional kinase involved in regulation of cell cycle (Cross et al., 1995; Ikeda et al., 1998; Wood et al., 2006) Last but not least, regulation of NF-κB

has also been shown to occur via a Btk/PI3K dependent pathway, through Akt and potentially via PLCγ2 (Dal Porto et al., 2004) This is based on studies in PI-3K

deficient B cells or using PI3K inhibitors, which show defects in NF-κB activation

(Petro and Khan, 2001; Saijo et al., 2002; Suzuki et al., 2003) The multiple functions

of NF-κB family of transcription factors play important role in B cell development,

proliferation and immunoglobulin class switching (Ruland and Mak, 2003b; Ruland and Mak, 2003a)

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1.3.2 Signaling via PLCγγγγ2 pathway

Upon BCR ligation, PLCγ2 is recruited to lipid raft via binding of its PH

domain to PI(3,4,5)P3 (produced by PI3K) or binding of its SH2 domain to BLNK, a critical adaptor protein in BCR signaling (Ishiai et al., 1999b; Marshall et al., 2000a) It was also reported that B cell adaptor protein, Bam32, plays a role in recruiting PLCγ2 to the membrane and hence leading to its activation (Marshall et al.,

phospho-2000a) However, among these, the association of PLCγ2 to BLNK appears to be

more critical in its activation since BLNK deficient B cells show almost complete failure of PLCγ2 membrane translocation and activation hence subsequent severe

defects in BCR signaling including ablated calcium (Ca2+) flux (Chiu et al., 2002; Ishiai et al., 1999a) BLNK is recruited to membrane via binding of its SH2 domain to non-ITAM phosphotyrosines of Ig signaling subunit and is subsequently phosphorylated by Syk upon BCR ligation (Engels et al., 2001; Kabak et al., 2002) Phospho-BLNK then serves as the binding site for SH2-domain containing proteins including PLCγ2 as well as Btk (Ishiai et al., 1999a; Kurosaki and Tsukada, 2000;

Wienands et al., 1998; Fu et al., 1998) Both Syk and Btk are essential kinases for optimal phosphorylation and activation of PLCγ2 (Chiu et al., 2002; Hashimoto et al.,

1999; Ishiai et al., 1999a) Therefore the role of BLNK as an adaptor protein to bring Syk and Btk to PLCγ2 for its activation is indispensable

Once activated, PLCγ2 cleaves the membrane associated phosphoinositide

PI(4,5)P2 into the second messengers I(1,4,5)P3 (IP3) and diacylglycerol (DAG) (Guo

et al., 2004; Marshall et al., 2000b; Rawlings, 1999; Su et al., 2002) IP3 triggers Ca2+

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mobilization from endoplasmic reticulum (ER) - the intracellular calcium stores This further triggers influx of Ca2+ from extracellular environment via triggering ion-gated calcium channel on the membrane Increased intracellular Ca2+ level will activate protein kinase Cs (PKCs) and Ca2+-calmodulin which in turn activate NF-κB and

Nuclear factor of activated T-cells (NFAT) transcription factors respectively (Dolmetsch et al., 1997; Saijo et al., 2002; Trushin et al., 1999) On the other hand, DAG activation leads to activation of other PKC isotypes The classical PKC isoforms ( , I, II and γ) are activated by both Ca2+ and DAG; the novel PKC isoforms (δ, ε, θ and η) are regulated by DAG but not by Ca2+; and the atypical PKC isoforms (ζ, τ and λ) are regulated by neither Ca2+ nor DAG (Guo et al., 2004) PKCs activated by DAG have been shown to play a role in regulating NF-κB and Mitogen-

activated protein (MAP) kinases (MAPK) family activities in particular c-Jun terminal kinase (JNK) and p38 MAPK (Guo et al., 2004) Both NF-κB and NFAT

N-play an essential role in regulating BCR-induced responses such as cell proliferation, differentiation and apoptosis (Antony et al., 2003; Serfling et al., 2004)

PLCγ2 deficient B cells show severe defects in BCR-induced activation of

JNK and p38 MAPK activation whereas activation of extracellular-signal-regulated kinase (Erk) is significantly impaired (Hashimoto et al., 1998)

1.3.3 Signaling via Ras/Raf/Erk pathway

Ras is a guanine nucleotide binding protein that is active when it is Guanosine triphosphate (GTP)-bound and inactive when it is Guanosine diphosphate (GDP)-

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bound Its activity is mainly controlled by 1) Guanine nucleotide exchange factors (GEFs) that promote GTP binding to Ras and 2) GTPases-activating proteins (GAPs) that activate RasGTPase to hydrolyse GTP to GDP (Genot and Cantrell, 2000) Ras pathway is activated upon BCR ligation as indicated by conversion from Ras-GDP to Ras-GTP state (Lazarus et al., 1993) Activation of Ras has been proposed to involve

a few pathways including first, via recruitment of SH2 domain-containing transforming protein C (Shc) to phosphorylated ITAM which leads to phosphorylation and activation of Shc by Syk Phospho-Shc binds to growth factor receptor-bound protein 2 (Grb2) which brings Son of Sevenless protein (SOS), a GEF

to Ras at the membrane fraction thereby activates Ras (D'Ambrosio et al., 1996; Nagai et al., 1995) However, subsequent studies have shown that Shc is dispensable and BLNK could play an analogous role in Ras activation (Hashimoto et al., 1998) Secondly, the phosphorylation of RasGAP (a negative regulator of Ras) by PKC leads

to a decrease in RasGAP activity hence an increase in Ras activity (Lazarus et al., 1993) Thirdly, via the activation of Ras guanine nucleotide releasing protein (RasGRP) by DAG which has been shown to positively regulates the activity of Ras (Coughlin et al., 2005; Ebinu et al., 1998)

Upon activation, Ras will activate Raf - downstreameffector kinase of Ras which leads to activation of Erk, a MAPK (Brummer et al., 2002; Tordai et al., 1994) Recent study also suggested that PI3K might also play a role in Ras and subsequently Erk activation (Jacob et al., 2002) This indicates the possibility that both Ras and PI3K may involve in a positive feedback loop to activate Erk

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The role of Erk in survival of other cell types has been established in previous studies (Ballif and Blenis, 2001) Inhibitor of Erk affect BCR-induced proliferation of mature B cells but does not affect the BCR-induced cell death in immature B cell lines (Richards et al., 2001) Following activation of Erk, transcription factors activated include Elk1 and c-Myc (Davis, 1995; Johnson et al., 1996)

1.3.4 Signaling via Vav/Rac pathway

Vav is a guanine nucleotide exchange factor (GEF) which shows specificity in promoting GTP-bound state of Rho family GTPase in particular Rac1 (Cantrell, 1998) Vav1 is preferentially expressed inhematopoietic lineage cells, whereas Vav2 and Vav3 are ubiqutinously expressed (Moores et al., 2000) Vav is recruited to phosphorylated CD19 and BLNK via its SH2 domain upon BCR ligation (Johmura et al., 2003; O'Rourke et al., 1998) This leads to activation of Rac and MAKPs - JNK and p38 (Hashimoto et al., 1998; Ishiai et al., 1999a; Salojin et al., 1999) Following that, JNK activates transcription factors such as c-Jun and ATF-2 whereas p38 MAPK activates transcription factors such as ATF-2 and MAX (Dong et al., 2002; Johnson and Lapadat, 2002)

Vav1 deficient B cells show defects in BCR-induced proliferation hence clearly indicating the role of Vav in BCR signaling (O'Rourke et al., 1998) However,

it is to be noted that JNK and p38 MAPK can also be regulated independently via PKC activation downstream of PLCγ2 as mentioned earlier

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Rac1, the main substrate of Vav also binds and activates phosphatidylinositol 4-phosphate (PIP)-5 kinase The main function of PIP-5 kinase is to phosphorylate PIP to PI(4,5)P2 which is the main substrate of PLCγ2 for production of IP3 and DAG and its downstream calcium mobilization (Cantrell, 1998) Therefore, Vav1-/- and Vav2-/- mice showed defects in BCR-induced calcium flux (Tedford et al., 2001) Recently, studies on Vav1/Vav2/Vav3 null mice show absence of functional T or B cells and complete failure in both T-dependent and T-independent B cell responses as well as BCR-induced calcium flux This indicates the role for the entire Vav protein family members in lymphocyte development and activation (Fujikawa et al., 2003)

Regulatory effect of Vav on Rac1 and RhoA affects the actin reorganization processes downstream of BCR ligation (Fischer et al., 1998; Holsinger et al., 1998) Phosphorylated BLNK also recruits Nck, an adaptor protein associated with cytoskeletal reorganization (Chiu et al., 2002; Mizuno et al., 2000) These actin reorganization processes upon BCR ligation will result in cellular morphogical changes as well as migration

1.4 Src family kinases

Association of Src family kinases (SFKs) with BCR complex upon ligation of BCR with antigen have been established since last decade The SFKs are nonreceptor protein tyrosine kinases that comprises Lyn, Src, Yes, Fgr, Fyn, Lck, Hck, Blk and Yrk (Brown and Cooper, 1996; Korade-Mirnics and Corey, 2000) There are a number of SFKs present in B lymphocytes: Lyn, Fyn, Blk, Hck, Fgr and Yrk (Lowell,

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2004) Among these, Lyn is known to be the primary SFKs required for the initiation

of BCR signaling (Gauld and Cambier, 2004) The common structural figures for SFKs are illustrated in Diagram 1.2 (Gauld and Cambier, 2004) Basically, they have

an amino (N)-terminal Src-homology (SH)-4 domain known to play an important role

in membrane localization of SFKs due to its acylation sites for both myristoylation and palmitoylation (processes critical for membrane localization) (Resh, 1999), except Blk which is only myristolated SH4 domain is followed by an unique domain and SH3 domain that mediates binding to proline rich regions (PXXP) of intracellular substrates (Gauld and Cambier, 2004; Lowell, 2004; Roskoski, Jr., 2004) Following SH3 is SH2 domain that mediates protein-protein interaction to tyrosine phosphorylated (especially YEEI motif) proteins (Songyang and Cantley, 1995; Tatosyan and Mizenina, 2000) At the carboxyl (C)-terminal there is protein kinase domain (SH1 domain) important for the catalytic kinase activity of SFKs which is to tyrosine phosphorylate downstream subtrates (Roskoski, Jr., 2004) Src family kinases have two important tyrosine phosphorylation sites: Tyrosine (Y)-416, an autophosphorylation site that is required for the kinase activation and a C-terminal tyrosine residue Y527 that negatively regulates the kinase (Roskoski, Jr., 2005)

Diagram 1.2: Structural features of Src family kinases Note that: the SH4 domain

of SFKs is for membrane targeting; the SH3 domain for binding to proline-rich containing proteins; the SH2 domain for binding to tyrosine-phosphorylated proteins;

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and the protein kinase domain (SH1) for catalytic kinase activity Two important tyrosine sites: Tyr 416 to enhance kinase activity while Tyr 527 mediates intramolecular binding to their own SH2 domain which keeps SFKs in a closed hence inactive conformation

Lyn is constitutively present in lipid raft but is kept inactive by tyrosine phosphorylation at its C-terminal Y527 which results in intramolecular binding to its own SH2 domain and hence maintaining the kinase in a closed or inactive conformation (Rajendran and Simons, 2005; Simons and Toomre, 2000) (Diagram 1.3) C-terminal Src tyrosine kinase (Csk) is known to be the kinase that phosphorylates the C-terminal Y527 of Lyn and thereby inhibits Lyn in resting B lymphocytes (Hata et al., 1994) Csk is maintained in the lipid rafts by binding to the phosphorylated Csk-binding protein (CBP) or also called phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), which resides in lipid raft together with SFKs at resting state (Brdicka et al., 2000; Kawabuchi et al., 2000) BCR ligation will first activates CD45, a phosphatase that dephosphorylates the C-terminal inhibitory tyrosine site Y527 in Lyn (Hermiston et al., 2003; Irie-Sasaki et al., 2003; Shrivastava et al., 2004) At the same time, BCR stimulation also triggers the dephosphorylation of CBP by a yet unknown mechanism Dephosphorylated CBP

no longer binds Csk and hence releases Csk to cytosol removing the inhibition of Lyn (Brdicka et al., 2000) This leads to the autophosphorylation of the Y416 residue in Lyn and thereby triggering its enzymatic activation On the other hand, BCR crosslinking will lead to translocation of BCR complex in lipid raft where the now activated Lyn can phosphorylate ITAM motifs in the cytoplasmic tails of Ig- and Ig-, the Ig complexed to BCR (DeFranco et al., 1995) Binding of the phosphorylated

It is to be noted that CD45 as a phosphatase can also dephosphorylate the stimulatory tyrosine residue (Y416) in Src family kinases leading to their inhibition Hence it is important for CD45 to be excluded from Src family kinases once it dephosphorylates the inhibitory tyrosine residue (Y527) in SFKs Indeed as shown in

T and B cells, CD45 is rapidly sequestered from immune synapse upon antigen receptors stimulation,

Diagram 1.3: Schematic illustration of activation and inactivation of Src kinases via phosphorylation and dephosphorylation Brieftly, at inactive state, Src is

phosphorylated at its C-terminal regulatory tyrosine site Y527 which leads to intramolecular binding to its own SH2 domain hence keeping Src in a closed or inactive conformation Upon stimulation, C-terminal tyrosine site is dephosphorylated (by CD45) and allows unclamping of the molecule which exposes its stimulatory tyrosine residue Y416 for autophosphorylation and hence activation of the kinase [Diagram obtained from (Roskoski, Jr., 2004)]

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Other than the well established role of Src kinases in BCR signal inititation it

is now known that Src family kinases, in particular Lyn can play both the negative and positive regulatory role in BCR signaling (Chan et al., 1997; Chan et al., 1998; Gauld and Cambier, 2004; Lowell, 2004; Nishizumi et al., 1998; Xu et al., 2005b) Lyn deficient mice are susceptible to autoimmune disease and Lyn-/- B cells were found to be hyperresponsive to BCR ligation despite defects in B cells development The details of negative regulatory role of Lyn will be discussed further in subsequent section

1.5 Adaptor proteins in lymphocyte signaling

Adaptor proteins are broadly defined as proteins that lack enzymatic or transcriptional activities but express various modular binding domains such as SH2, SH3, Phospho-tyrosine binding (PTB) and PH domains or tyrosine phosphorylation sites (Kurosaki, 2002; Leo and Schraven, 2001) These domains or motifs are known

to mediate protein-protein or protein-lipid interactions which are important for signal transduction and integration (Pawson and Scott, 1997) For instance, SH2 and PTB domains are known to recognize proteins with tyrosine phosphorylated sites (Schlessinger and Lemmon, 2003) While SH3 domains bind to proline-rich regions containing proteins (Gao et al., 2006; Macias et al., 2002) PH domains mediate protein-lipid interactions as they recognize phosphoinositides (Lemmon, 2007) Recent studies also show the ability of PH domains to mediate protein-protein interactions such as PH domain of Etk, a member of Btk family kinase, is able to bind

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the FERM domain of focal adhesion kinase (FAK) (Chen et al., 2001) Recently the definition of adaptor proteins has broadened to include some of the proteins that have enzymatic activity For example, Lyn, a Src family kinase, which has both the SH2 and SH3 domains that can facilitate protein-protein interactions, is now considered as

an adaptor protein as well (Kurosaki, 2002)

Adaptor proteins play an important role in mediating intracellular signaling by facilitating intermolecular interactions in a spatial and temporal manner In the immune system, they play critical roles in lymphocyte activation by assembling signaling complexes at the activated plasma membrane or lipid raft (Veillette, 2004; Jordan et al., 2003; Kurosaki, 2002) A list of adaptor proteins identified in lymphocytes signaling is shown in Diagram 1.4 (Leo and Schraven, 2001) In B cells, several adaptor proteins such as BLNK or SLP-65 have been shown to play important roles in B cell development or activation (Horejsi, 2004; Janssen and Zhang, 2003; Fu

et al., 1998) The role of BLNK has been widely established as an adaptor protein that couples Syk and Btk with PLCγ2 upon BCR ligation which leads to downstream

calcium flux and IP3 production (Kabak et al., 2002)

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Diagram 1.4: A list of adaptor proteins identified in lymphocytes Note the

different domains and motifs important for mediating protein-protein as well as

protein-lipid interactions Diagram obtained from (Leo and Schraven, 2001)

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In recent years, the role of negative adaptor proteins took centre stage as many adaptor proteins such as Dok-3 and CBP were found to play essential roles in negative regulation of antigen receptor signaling in lymphocytes (Yamasaki and Saito, 2004) Most of these inhibitory adaptors function by recruiting inhibitory effectors to the vicinity of positive regulators e.g Cbp recuits Csk (Xu et al., 2005a) whereas Downstream of tyrosine kinases (Dok)-3 recruits SH2-containing inositol 5’-phosphatase (SHIP) (Robson et al., 2004) These inhibitory effectors are mostly the phosphatases which will dephosphorylate and deactivate the positive regulators such

as the deactivation of Lyn by Csk (Hata et al., 1994)

1.5.1 Bam32

Bam32 has the protein size of 32 kD as the name suggests (Dowler et al., 1999; Marshall et al., 2000a; Rao et al., 1999) and it is also called Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides (DAPP1) or 3’-Phosphoinositide- Interacting SRC Homology-containing protein (PHISH) It was identified by four

independent groups using subtractive hybridization PCR (Marshall et al., 2000a), purification based on phosphoinositide binding (Rao et al., 1999) and EST database searching for PH domain proteins containing the 3-phophoinositide-binding motif (Dowler et al., 1999; Isakoff et al., 1998)

Bam32 is an adaptor protein restricted to hematopoietic cells and is highly expressed in germinal center (GC) B cells (Niiro et al., 2002) It has a N-terminal SH2 domain which is known to associate with PLCγ2 and a C-terminal PH domain

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with high affinity for PI(3,4,5)P3 and PI(3,4)P2 (Marshall et al., 2000a) It is phosphorylated at its single centrally located tyrosine phosphorylation site Y139 upon BCR stimulation (Anderson et al., 2000) (Diagram 1.5) The phosphorylation of Y139 on Bam32 is believed to be mediated by Src family kinases based on the fact that its phosphorylation can be blocked by inhibitors of Src family kinases (Dowler et al., 2000b; Niiro et al., 2004) and a Platelet-derived growth factor (PDGF) receptor with mutations in Src-kinase binding sites (Dowler et al., 2000b) Bam32 phosphorylation is defective in Lyn deficient but not in Btk deficient DT40 chicken B cells (Dowler et al., 2000b), indicating the role of Lyn as the Src kinase responsible for phosphorylating Bam32 upon BCR ligation

Diagram 1.5 Schematic illustration of the major structural features of Bam32

Bam32 has a N-terminal SH2 domain which is known to associate with PLCγ2 and a

C-terminal PH domain with high affinity for PI(3,4,5)P3 and PI(3,4)P2

Bam32 is recruited to the plasma membrane via its PH domain in a PI3-K dependent manner upon BCR ligation and accumulated within F-actin-rich membrane ruffles (Anderson et al., 2000; Allam et al., 2004) However, PH domain of Bam32 shows preferential binding to PI(3,4)P2 over PIP3 in competitive binding studies (Ferguson et al., 2000) Hence in contrast to Btk, Bam32 as well as its homologues -Tandem PH domain-containing proteins (TAPPs) (as described in more details later) show delayed and sustained membrane recruitment upon BCR ligation consistent

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with the production of PI(3,4)P2 (Marshall et al., 2002) The production of PI(3,4)P2

from PIP3 is believed to be the action of SHIP which is activated later in order to downregulate BCR signaling after its stimulation (Brauweiler et al., 2000; Damen et al., 1996)

Bam32 is also linked to cytoskeletal rearrangement which leads to membrane ruffling and receptor internalization (Niiro et al., 2004) Upon BCR ligation, it induces F-actin accumulation by regulating GTPase Rac1 activity, a key molecule controlling actin remodeling in leukocytes (Diagram 1.6) The phosphorylation of Y139 on Bam32 is suggested to be a key factor in these functions (Allam et al., 2004)

Studies on Bam32 knockout mice show that Bam32 plays an important role in

T cell-independent type II (TI-2) immune responses Bam32-/- B cells fail to undergo class switching to produce IgG3 antibodies and hence are more susceptible to infection by encapsulated bacteria such as Streptococcus pneumoniae BCR-induced proliferation and activation of JNK and ERK are also impaired in Bam32-/- B cells (Han et al., 2003; Fournier et al., 2003) The mechanism of how Bam32 regulates BCR signaling is still largely unknown A previous report claims that Bam32 interacts with the hematopoietic progenitor kinase-1 (HPK1) thereby regulating BCR signaling pathways such as PLCγ2/Calcium/NFAT and MAPK, in particular JNK and

ERK (Niiro and Clark, 2003)

The mechanism of action of Bam32 is illustrated in Diagram 1.6 obtained from a recent review by Allam A et al (Allam and Marshall, 2005)

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Diagram 1.6: Model of Bam32 functions in B cell activation Bam32 is first

recruited to the membrane upon BCR ligation via binding of its PH domain to PI(3,4)P2 or PIP3 and be tyrosine-phosphorylated by Src family kinase (most probably Lyn) Activated Bam32 then activates Rac1 which leads to actin reorganization hence membrane ruffling and BCR internalization as well as activation of JNK Bam32 can bind to HPK1 which then leads to downstream activation of MEKK1, JNK and Erk Bam32 can also induce NF-AT activation via PLCγ2/Ca2+ pathway or HPK1 Cumulatively, Bam32 regulates TI-II immune response and B cells proliferation Single arrows = potential direct interactions; double arrows = indirect interactions that likely involve intermediate steps and; question marks = areas requiring further confirmation or where there are conflicting data Diagram obtained from (Allam and Marshall, 2005)

1.5.2 TAPP 1 & 2

Bam32 belongs to a Tandem PH domain-containing proteins (TAPPs) family

of adaptor proteins which includes two other members TAPP1 and TAPP2 (Allam and Marshall, 2005; Marshall et al., 2002) As compared to Bam32 which is preferentially expressed in hematopoietic cells and tissues, TAPPs are ubiquitously