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.. Evidences obtained from physiological studies of Dok-1 and 2 single and double kno

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

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Table of Contents

List of Schematic Diagrams and Tables xiii

Chapter 1 Introduction

1.2.1 Subpopulations of B lymphocytes 4

1.3 B cell receptor signal transduction 7

1.3.2 The Ras signaling pathway 12

1.4 Negative regulators of B cell receptor signaling 14

1.4.2 SH2-containing inositol 5’-phosphatase-1

1.4.4 C-terminal Src tyrosine kinase (Csk) 23 1.4.5 Downstream of tyrosine kinases (Dok) 23

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1.4.5.3 Dok-4, 5 and 6 39

1.6 Rationale and aims of this project 45 Chapter 2 Materials and Methods

2.3.2 Mouse tail genomic DNA prep 52

2.3.8 Elution of DNA from agarose gel 56

2.3.11 Dephosphorylation of plasmid DNA 57

2.3.13 Preparation of DH5 α competent cells 58 2.3.14 Transformation of DH5 α 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

2.4 Mammalian cell culture methodology 60

2.4.3 Purification of bone marrow cells 61

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2.5.2 Cloning of targeting construct 64 2.5.3 Culturing of mouse embryonic fibroblasts 64

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-

2.5.8 Expanding ES clones from 48-well to 24-well

2.5.9 Preparation of genomic DNA from ES cells 67

2.5.11 Microinjection of positive ES clones 67 2.6 Molecular and cellular immunology methodology 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

2.7.2 Immunoprecipitation, western blot and

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

3.2 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

3.3.2 Dok-3 is not required for B cell development

in the peripheral immune tissues 84

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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

3.4.3 Loss of dok-3 does not affect T-cell dependent

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

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

3.7 Conclusion and future directions 111

3.7.1 Role of Dok-3 in Fc γRIIB 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

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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,

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

N-terminus Pleckstrin (PH) and Phospho-tyrosine binding (PTB) domains as well as

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

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(MAPK), in particularly Extracellular signal-regulated kinases (Erk) Dok-1 and 2 may

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

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

peripheral immune organs Detailed FACS analysis of all relevant leukocyte populations

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

in contrast to wildtype littermates Mutants showed elevated antigen-specific serum

primary and secondary responses when challenged by a T cell-dependent (TD) antigen

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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

alone or together with CD40 ligand (CD40L)

To further dissect the molecular mechanisms pertaining to the

BCR 3 major MAPK pathways, ERK, c-Jun N-terminal kinase (JNK) and p38, are

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

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

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

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inhibition of calcium flux Indeed there was elevated magnitude of calcium flux in Dok-3

phosphorylation of SHIP-1 but not its localization to the plasma membrane We note that

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)

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Abbreviations

Ig Immunoglobulin

IL Interleukin

x

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

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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

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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

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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

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Chapter 1 Introduction