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

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.


TABLE OF CONTENTS
SUMMARY i
ABBREVIATIONS iii
LIST OF SCHEMATIC DIAGRAMS AND TABLES v
LIST OF FIGURES vi
LIST OF PUBLICATIONS ix
CHAPTER 1: INTRODUCTION
1.1 The immune system 1
1.2 Innate and adaptive immunity 1
1.3 B cell antigen receptor signaling pathways 3
1.3.1 Signaling via PI3-K pathway 7
1.3.2 Signaling via PLCγ2 pathway 9
1.3.3 Signaling via Ras/Raf/Erk pathway 10
1.3.4 Signaling via Vav/Rac pathway 12
1.4 Src family kinases 13
1.5 Adaptor proteins in lymphocyte signaling 17
1.5.1 Bam32 20
1.5.2 TAPP 1 & 2 23
1.6 Negative regulatory pathways of B cell antigen receptor signaling 26
1.6.1 Protein tyrosine phosphatases (PTPs) 26
1.6.1.1 SHP-1 26
1.6.1.2 PEP 28
1.6.2 Lipid Phophatases 29
1.6.2.1 SHIP-1 29

1.6.2.2 PTEN 31
1.6.3 Protein tyrosine kinases 32
1.6.3.1 Csk 32
1.6.3.2 Lyn 33
1.6.4 Cbl Family of Ubiquitin Ligases 35
1.6.4.1 C-Cbl 35
1.6.4.2 Cbl-b 36
1.7 Paxillin superfamily members 36
1.7.1 Paxillin 39
1.7.2 Hic-5 40
1.7.3 Leupaxin 42
1.8 Rationale and aims of this project 43




CHAPTER 2: MATERIAL AND METHODS

2.1 List of antibodies for Immuno-fluorescence, BCR stimulation, Immuno-
precipitation and Immuno-blotting 45
2.2 List of primers 46
2.3 Molecular cloning methodology 47
2.3.1 Buffers and solutions 47
2.3.2 Plasmids DNA constructs 50
2.3.3 Extraction of RNA 50
2.3.4 First strand cDNA synthesis 51
2.3.5 Polymerase chain reaction 52
2.3.6 DNA sequencing 53
2.3.7 Restriction digestion of DNA 54
2.3.8 Agarose gel electroporesis 55

2.3.9 Elution of DNA from agarose gel 55
2.3.10 Dephosphorylation of plasmid DNA 56
2.3.11 Ligation of DNA 56
2.3.12 Preparation of DH5α competent cells 57
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
2.4 Mammalian cell culture methodology 59
2.4.1 Cell culture media 59
2.4.2 Purification of splenic B cells 60
2.4.3 Transfection of HEK 293T cells 61
2.4.4 Transfection of A20 B cells 62
2.4.5 Stimulation of A20/ BJAB cells 62
2.5 Molecular and cellular immunology methodology 62
2.5.1 Flow cytometry 62
2.5.2 BCR-induced IL-2 production 63
2.5.3 BCR-induced activation of IL-2 promoter 63
2.5.4 Confocal microscopy 64
2.6 Protein methodology 65
2.6.1 Buffers and solutions 65
2.6.2 Immunoprecipitation 66
2.6.3 Western blotting 67
2.6.4 Isolation of membrane fraction 68

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

3.1 Introduction 69
3.2 Results 72
3.2.1 Yeast-two-Hybrid using B cells adaptor protein, Bam32 as a bait 72
3.2.2 Interaction of Bam32 with Lyn 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
and paxillin 75
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
cells. 85
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
3.3 Discussion 97
3.4 Future directions 102
3.5 Conclusion 104

CHAPTER 4: THE ROLE OF LEUPAXIN IN B CELL RECEPTOR
SIGNALING.

4.1 Introduction 105
4.2 Results 107
4.2.1 Sequence consensus between human and mouse leupaxin 107
4.2.2 Leupaxin is tyrosine phosphorylated upon BCR ligation in human
BJAB B cells 109
4.2.3 Leupaxin is recruited to the plasma membrane upon BCR ligation in
human BJAB B cells 112
4.2.4 Leupaxin interacts with Lyn 113
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
B cells 123
4.2.8 Leupaxin inhibits IL-2 production in A20 B cells 128
4.2.9 Tyrosine 72 of leupaxin is important for its inhibitory function 132
4.3 Discussion 137
4.4 Future directions 141
4.5 Conclusion 144

CONCLUSION 145

LIST OF REFERENCES 146

PUBLICATIONS


i

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

ii

specifically in BCR-induced phosphorylation of p38 MAPK. Despite the current
findings, the detailed mechanism of the function of Hic-5 in BCR signaling remains
to be elucidated.
The role of another member of paxillin superfamily proteins- leupaxin was
explored in our current project as well. First we showed that leupaxin (LPXN) is
tyrosine-phosphorylated and recruited to the plasma membrane of human BJAB
lymphoma cells upon BCR stimulation, and interacts with Lyn in a BCR-inducible
manner. LPXN contains four leucine-rich sequences termed LD motifs and serial
truncation and specific domain deletion of LPXN indicated that its LD3 was involved
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.


iii
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
GDP Guanosine diphosphate
Grb2 Growth factor receptor-bound protein 2
GTP Guanosine triphosphate
HA Haemagglutinin
HPK1 Hematopoietic progenitor kinase-1
I(1,3,4,5)P
4
1,3,4,5-tetrakisphosphates
Ig Immunoglobulin

IL Interleukin
IP
3
Inositol 3,4,5-triphosphate
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)
LPXN Leupaxin
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)P
2
Phosphatidylinositol 3,4-bisphosphate
PI(3,4,5)P
3
Phosphatidylinositol 3,4,5-triphosphate
PI3-K Phosphatidylinositol 3-kinase
PI(4,5)P
2
Phosphatidylinositol 4,5-bisphosphate
PCR Polymerase chain reaction
PH Pleckstrin homology
PKB Protein kinase B

PKC Protein kinase C
PLCγ2 Phospholipase C gamma 2
PTB Phospho-tyrosine binding

iv

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


v

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

vi

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


vii

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-κ
κκ
κB activation


viii
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

1

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


2

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


4

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:


6

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 IP
3
and Ca
2+
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).





7

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)P

2
)] to Phosphatidylinositol 3,4,5-triphophate [PI(3,4,5)P
3
]
(Cantrell, 2002). PI(3,4,5)P
3
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)P
3
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.


8

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)P
3
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).