Elucidating the role of dok 3 in b cell receptor signaling using gene knockout mice

ELUCIDATING THE ROLE OF DOK-3
IN B CELL RECEPTOR SIGNALING USING
GENE KNOCKOUT MICE






NG CHEE HOE
(BSc. Biochemistry (Hons.), NUS)








A THESIS SUBMITTED FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY

INSTITUTE OF MOLECULAR AND CELL BIOLOGY

NATIONAL UNIVERSITY OF SINGAPORE



Acknowledgements


I would like to take this opportunity to show my appreciation and thank a few
colleagues in IMCB which make this journey possible. First and foremost, to my
supervisor A/P Lam Kong Peng for his patience, valuable suggestions and guidance
during the course of this project. Heartfelt thanks are also given to members of the
supervisory committee, Dr Walter Hunizker and Dr Li Bao Jie for their useful
suggestions and meaningful discussion during the committee meetings.
I would like to express my gratitude to my colleagues and collaborators, Dr Wong
Siew Cheng, Dr Xu Shengli and Dr Ng Lai Guan. I have learnt a lot from all of you
during our collaborations in the B7-H2, Dok-3 and B-1 cell projects. To all my fellow
colleagues from the LKP lab, past and present, Joy, Weng Keong, Kar Wai, Andy,
Valerie, Ann Teck, Jianxin and Koon Guan for sharing reagents and meaningful
discussion about science and the companionship for the past 6 years. Weng Keong,
thanks for managing our laboratory and keeping it a conducive work place to carry out
our experiment.
Sincere thanks to Guo Ke, Li Jie and Bin Qi from the Histology Services for their
assistance and imparting their knowledge on tissue sectioning and immunocytochemistry;
to all the staffs from the Biological Resource Centre who had help in the microinjection
of Dok-3 ES cells, maintaining and changing the many cages of mice, and the staffs from
DNA Sequencing Facility for their efforts in DNA sequencing.
Last but not least, I am in debt to my family and my girlfriend, Joyce, for their
strong morale support, understanding and love. This would be impossible without you.

i
Table of Contents

Acknowledgements i

Table of Contents ii

Summary vi

Abbreviations x

List of Figures xii

List of Schematic Diagrams and Tables xiii

List of Publications xiv

Chapter 1 Introduction

1.1 Innate and adaptive immunity 1

1.2 B lymphocytes 2

1.2.1 Subpopulations of B lymphocytes 4
1.2.2 B cell development 5

1.3 B cell receptor signal transduction 7

1.3.1 The PLCγ2 pathway 11
1.3.2 The Ras signaling pathway 12

1.3.3 The Rho GTPase pathway 12
1.3.4 The PI3-K pathway 14

1.4 Negative regulators of B cell receptor signaling 14

1.4.1 FcγRIIB receptor 16
1.4.2 SH2-containing inositol 5’-phosphatase-1
(SHIP-1) 18
1.4.3 Lyn tyrosine kinase 21
1.4.4 C-terminal Src tyrosine kinase (Csk) 23
1.4.5 Downstream of tyrosine kinases (Dok) 23
1.4.5.1 Dok-1 and Dok-2 27
1.4.5.2 Dok-3 33
ii
1.4.5.3 Dok-4, 5 and 6 39
1.4.5.4 Dok-7 41

1.5 Gene targeting 42

1.6 Rationale and aims of this project 45

Chapter 2 Materials and Methods

2.1 List of antibodies 47

2.2 List of primers 48

2.3 Molecular cloning methodology 48

2.3.1 Buffers and solutions 48

2.3.2 Mouse tail genomic DNA prep 52
2.3.3 Genotyping of Dok-3 mice 53
2.3.4 Polymerase chain reaction 54
2.3.5 DNA sequencing 54
2.3.6 Restriction digestion of DNA 55
2.3.7 Agarose gel electrophoresis 56
2.3.8 Elution of DNA from agarose gel 56
2.3.9 Southern hybridization 56
2.3.10 Probe labeling 57
2.3.11 Dephosphorylation of plasmid DNA 57
2.3.12 Ligation of DNA 58
2.3.13 Preparation of DH5α competent cells 58
2.3.14 Transformation of DH5α by heat shock method 59
2.3.15 Bacterial DNA mini-prep by alkaline lysis 59
2.3.16 Bacterial maxi-prep using Qiagen Maxi-prep
columns 60

2.4 Mammalian cell culture methodology 60

2.4.1 Cell culture media 60
2.4.2 Purification of splenic B cells 61
2.4.3 Purification of bone marrow cells 61
2.4.4 Purification of thymocytes 62
2.4.5 Purification of lymph node cells 62
2.4.6 Proliferation assay 62

2.5 Gene targeting methodology 63

2.5.1 Buffers and solutions 63
iii

2.5.2 Cloning of targeting construct 64
2.5.3 Culturing of mouse embryonic fibroblasts 64
2.5.4 Culturing of ES cells 65
2.5.5 Preparation of DNA for transfection 65
2.5.6 Transfection and selection of ES cells 65
2.5.7 Picking and freezing down of ES clones in 48-
well plate 66
2.5.8 Expanding ES clones from 48-well to 24-well
plate 66
2.5.9 Preparation of genomic DNA from ES cells 67
2.5.10 Screening of positive ES clones 67
2.5.11 Microinjection of positive ES clones 67

2.6 Molecular and cellular immunology methodology 68

2.6.1 Buffers and solutions 68
2.6.2 Flow cytometry 68
2.6.3 Enzyme-linked immuno-sorbent assay (ELISA) 68
2.6.4 Preparation of Alum-precipitated antigen 69
2.6.5 T-independent and T-dependent in vivo
immunizations 69
2.6.6 Immunocytochemistry and histology 70
2.6.7 Intracellular calcium analysis 70

2.7 Protein methodology 70

2.7.1 Buffers and solutions 70
2.7.2 Immunoprecipitation, western blot and
antibodies 71
2.7.3 Isolation of membrane fraction 72

2.7.4 Nuclear fractionation and gel shift assay 72

Chapter 3 Dok-3: An inhibitory adaptor regulating B cell receptor
signaling

3.1 Introduction 75

3.2 Generation of Dok-3 deficient mice 76

3.3 Studying the role of Dok-3 in B cell development 81

3.3.1 Dok-3 is dispensable for B cell development in
the bone marrow 81
3.3.2 Dok-3 is not required for B cell development
in the peripheral immune tissues 84
iv
3.4 Role of Dok-3 in humoral immune responses 87

3.4.1 Dok-3
-/-
mice exhibit elevated basal serum IgM 87

3.4.2 Dok-3
-/-
mice are hyper-responsive towards T-
independent antigens 90
3.4.3 Loss of dok-3 does not affect T-cell dependent
humoral immune responses 93

3.4.4 Aged Dok-3

-/-
mice did not succumb to
autoimmunity 95

3.5 Role of Dok-3 in B cell receptor activation 97

3.5.1 Dok-3 negatively regulates BCR-stimulated
proliferation 97

3.5.2 Enhanced calcium signaling in Dok-3
-/-
B cells 99

3.5.3 Enhancement of NF-κB activation in BCR-
stimulated Dok-3
-/-
B cells 101

3.5.4 Enhanced activation of JNK and p38 MAPK in
BCR-stimulated Dok-3
-/-
B cells 103

3.5.5 Dok-3-deficiency impairs SHIP-1 activation
but not SHIP-1 localization 105

3.6 Discussion 108

3.7 Conclusion and future directions 111
3.7.1 Role of Dok-3 in FcγRIIB signaling 112

3.7.2 Role of Dok-3 in TLR signaling 113
3.7.3 Interaction of Dok-3 with G3BP-1 in the
immune system 114

References 118
Publications 133

v
Summary

Adaptor or docking proteins possess multiple modular domains responsible for
recruiting signaling proteins to activated receptors, nucleating intermolecular complexes
and positively or negatively modulating effector protein activity by inducing
conformational changes or phosphorylation/dephosphorylation. One emerging class of
adaptors known as Dok (downstream of tyrosine kinases) consists of 7 members,
including Dok-1 (p62
dok
), Dok-2 [p56
dok-2
, Dok-Related (Dok-R) or Interleukin Four
Receptor Interacting Protein (FRIP)], Dok-3 [Dok-Liked (Dok-L)], Dok-4 [Insulin
Receptor Substrate-5 (IRS-5)], Dok-5 (IRS-6), Dok-6 and Dok-7. These proteins though
having different expression profiles, are structurally similar,

containing tandem N-
terminus Pleckstrin (PH) and Phospho-tyrosine binding (PTB) domains as well as
multiple potential Src-homology 2 (SH2)

and SH3 binding sites at their C-terminus,
depicted as tyrosine residues phosphorylated during activation.

Three Dok family members are expressed mainly in hematopoietic cells, namely
Dok-1, 2 and 3. Dok-1 and 2 are adaptor molecules originally identified as Bcr-Abl
substrates that are hyperphosphorylated in Chronic Myeloid Leukemia (CML) patients.
They are implicated in tumorgenesis, cell proliferation and cell migration. Evidences
obtained from physiological studies of Dok-1 and 2 single and double knockout mice
strongly indicate the importance of these adaptors in regulating FcγRIIB-dependent
proliferation, Bcr-Abl-induced transformation, homeostasis of myeloid cells,
leukemogenesis, lipopolysaccharide (LPS) endotoxin shock and T cell receptor signaling.
Both adaptors act as negative regulators of Ras and mitogen activated protein kinase
vi
(MAPK), in particularly Extracellular signal-regulated kinases (Erk). Dok-1 and 2 may
exert their inhibitory effects by recruitment

of Ras GTPase Activating Protein (RasGAP),
a negative regulator of Ras signaling.

Overexpression of Dok-3 in cell lines showed that
Dok-3 is a negative regulator of B cell receptor (BCR) and v-Abl signaling possibly via
recruiting SH2-containing inositol phosphatase 1 (SHIP-1), Growth factor receptor-
bound protein 2 (Grb2)

and C-terminal Src tyrosine kinase (Csk). To date, the
physiological function of Dok-3 is still unclear. Here we have generated the Dok-3
deficient mice and focus our current study on B cell development, function and activation
since Dok-3 was reported to be mainly expressed in B cells.
We found that Dok-3
-/-
mice have normal B cell population in the central and
peripheral immune organs. Detailed FACS analysis of all relevant leukocyte populations
obtained from Dok-3

-/-
mice did not reveal any change in their fractions, percentage or
absolute numbers compared to wildtype littermate control. Thus Dok-3 does not play an
essential role in the development of B cells.
In vivo, these mutant mice exhibit elevated basal serum IgM but normal IgG1,
IgG2a, IgG2b and IgG3. Next to determine Dok-3 functions in humoral immune
responses, we challenged wildtype and Dok-3
-/-
mice with both type I and II T-
independent (TI) antigens. In both studies, challenged Dok-3
-/-
mice are hyper-responsive
in contrast to wildtype littermates. Mutants showed elevated antigen-specific serum
immunoglobulins, IgM and IgG3. Interestingly Dok-3
-/-
mice showed comparable
primary and secondary responses when challenged by a T cell-dependent (TD) antigen.
Dok-3
-/-
mice secreted comparable amounts of antigen-specific sera immunoglobulins as
vii
wildtype controls. We sectioned spleens obtained from antigen-challenged mice and
immunostained with peanut agglutinin (PNA), a germinal centre-specific marker, and did
not find profound difference between their morphology.
Mature B cells undergo rapid clonal expansion upon activation by antigens
through their BCR. To assess Dok-3 function in B cell activation, we collected splenic B
cells from both wildtype and Dok-3
-/-
mice, stimulated them in culture through the BCR
and measured their capacity to proliferate. Consistent with the in vivo results, Dok-3

-/-
B
cells hyper-proliferated in response to B cell receptor stimulated with ligand IgM F(ab’)
2

alone or together with CD40 ligand (CD40L).
To further dissect the molecular mechanisms pertaining to the hyper-
responsiveness in Dok-3
-/-
B cells, we studied intracellular events downstream of the
BCR. 3 major MAPK pathways, ERK, c-Jun N-terminal kinase (JNK) and p38, are
important signal transduction cascades. Dok-3
-/-
cells showed normal ERK
phosphorylation which is accompanied by enhanced phosphorylation of JNK and p38.
Other hallmark events that are displayed by an activated B cell include activation of
transcription factors such as NF-κB. We showed that Dok-3
-/-
B cells exhibited increased
IκBα degradation accompanied by enhanced NF-κB DNA binding. SHIP-1 is an
important candidate to investigate as it is a pivotal negative regulator in B cell and was
shown to bind Dok-3 upon B cell activation. Active SHIP-1 is phosphorylated at Tyr-914
and Tyr-1020. We found that Dok-3
-/-
B cells, SHIP-1 was not activated optimally upon
BCR stimulation, with less sustained phosphorylation at Tyr-1020. This critical tyrosine
when phosphorylated can bind to Shc and mutagenesis of the tyrosine can abolish SHIP-1
viii
inhibition of calcium flux. Indeed there was elevated magnitude of calcium flux in Dok-3
-

/-
B cells stimulated through BCR. Interestingly, loss of Dok-3 only impaired the
phosphorylation of SHIP-1 but not its localization to the plasma membrane. We note that
phenotype of Dok-3
-/-
mice is quite identical to that of SHIP-1
-/-
mice. These data are
consistent with the postulated role of Dok-3 as an inhibitory signaling molecule that
possibly acts through SHIP-1 to attenuate BCR signaling. Indeed, a recent report
supported my thesis finding in that its negative role in regulation of cellular calcium flux
was highlighted using Dok-3 deficiency DT40 chicken B cell line (Stork et al, 2007).














ix
Abbreviations
Abl Abelson murine leukemia
AChR Acetylcholine receptor

BCR B cell receptor
BLNK B cell linker protein
BMDM Bone marrow-derived macrophage
Btk Bruton’s tyrosine kinase
CML Chronic Myeloid Leukemia
Csk C-terminal Src tyrosine kinase
DAG Diacylglycerol
DNA Deoxyribonucleic acid
Dok Downstream of tyrosine kinases
ERK Extracellular-signal-regulated kinase
ES Embryonic stem
FACS Florescence activated cell sorting
FITC Fluorescein isothiocyanate
FRIP Interleukin Four Receptor Interacting Protein
G3BP RasGAP SH3 domain binding protein
G418 Geneticin
GANC Gancyclovir
Grb2 Growth factor receptor-bound protein 2
HA Haemagglutinin
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
LIF Leukemia inhibitory factor
LPS Lipopolysaccharide
Lyn Lck/yes-related novel tyrosine kinase
MAPK Mitogen activated protein kinase
MAPKK Mitogen activated protein kinase kinase
MAPKKK Mitogen activated protein kinase kinase kinase
MHC Major histocompatibility complex
MCSF Macrophage colony-stimulating factor
MuSK Muscle-specific receptor kinase
NF-κB Nuclear factor κB
PAMP Pathogen-associated molecular pattern
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
x
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
PTEN Phosphatase and tensin homolog deleted on chromosome 10

PTK Protein tyrosine kinase
RasGAP Ras GTPase activating protein
SH2 Src-homology 2
SH3 Src-homology 3
SHIP-1 SH2-containing inositol 5’-phosphatase-1
SHP-1 SH2-domain containing protein tyrosine phosphatase-1
SOS Son of sevenless
Syk Spleen-associated tyrosine kinase
TCR T cell receptor
TLR Toll-like receptor
TNF-α Tumor necrosis factor alpha

xi
List of Figures
Figure 1.1 mRNA expression of Dok-1, 2 and 3 in various mouse tissues and cell
lines

Figure 1.2 Expression of Dok-1, 2 and 3 genes in hematopoietic cells

Figure 3.1 Dok-3 gene knockout strategy

Figure 3.2 Inactivation of dok-3 gene locus

Figure 3.3 B cell development is normal in Dok-3
-/-
bone marrow

Figure 3.4 Normal B cell development in peripheral immune tissues of Dok-3
-/-

mice

Figure 3.5 Measurement of basal serum antibodies in wild-type and Dok-3
-/-
mice

Figure 3.6 Humoral immune responses to T-independent type I and II antigen

Figure 3.7 Humoral immune response to T-dependent antigen

Figure 3.8 Dok-3 deficiency alone is not sufficient to give rise to autoimmunity

Figure 3.9 Hyperproliferation of Dok-3
-/-
B cells in response to BCR stimulation

Figure 3.10 Enhanced calcium signaling in Dok-3
-/-
B cells

Figure 3.11 Enhanced NF-κB activation in BCR-stimulated Dok-3
-/-
B cells

Figure 3.12 Enhanced activation of JNK and p38 MAPK signaling in BCR-
stimulated Dok-3
-/-
B cells

Figure 3.13 Dok-3 deficiency leads to less sustained SHIP-1 activation after BCR
stimulation

Figure 3.14 Interactions of Dok-1 and Dok-3 with G3BP-1 and G3BP-2

xii
List of Schematic Diagrams and Tables

Diagram 1 B cell receptor signaling pathway

Diagram 2 SHIP-1 mediated inhibition of cellular activation

Diagram 3 Structure and domains of Dok family members

Diagram 4 Amino acid sequences of chicken, mouse and human Dok-3 orthologs
aligned using ClusterW algorithms

Diagram 5 Domains of Dok-3 and its binding partner

Diagram 6 T-independent and T-dependent immunization regime

Diagram 7 Structure of G3BP-1 and 2

Table 1 Summary of Dok family members

Table 2 Enumeration of total and lymphocytes populations in various tissues
of wild-type and Dok-3
-/-
mice

Table 3 Enumeration of total cells and B220
+
IgM
+
and CD5

+
lymphocyte
populations in various tissues of wild-type and Dok-3
-/-
mice













xiii
List of Publications

Ng CH, Xu S, Lam KP
Dok-3 plays a non-redundant role in negative regulation of B cell activation.
Blood. 2007 Jul 1:110(1):259-266. (Inside Blood in Blood 110(1):3-4)

Ng LG, Ng CH, Woehl B, Sutherland AP, Huo J, Xu S, Mackay F, Lam KP.
BAFF costimulation of Toll-like receptor-activated B-1 cells.
Eur J Immunol. 2006 Jul;36(7):1837-46.

Wong SC, Oh E, Ng CH, Lam KP.

Impaired germinal center formation and recall T-cell-dependent immune responses in
mice lacking the costimulatory ligand B7-H2.
Blood. 2003 Aug 15;102(4):1381-8.

xiv


















Chapter 1

Introduction