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 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 Introduction 1.1 Innate and adaptive immunity 1.2 B lymphocytes 1.2.1 Subpopulations of B lymphocytes 1.2.2 B cell development B cell receptor signal transduction 1.3.1 The PLCγ2 pathway 1.3.2 The Ras signaling pathway 1.3.3 The Rho GTPase pathway 1.3.4 The PI3-K pathway 11 12 12 14 Negative regulators of B cell receptor signaling 14 1.4.1 FcγRIIB receptor 1.4.2 SH2-containing inositol 5’-phosphatase-1 (SHIP-1) 1.4.3 Lyn tyrosine kinase 1.4.4 C-terminal Src tyrosine kinase (Csk) 1.4.5 Downstream of tyrosine kinases (Dok) 1.4.5.1 Dok-1 and Dok-2 1.4.5.2 Dok-3 16 1.3 1.4 18 21 23 23 27 33 ii 1.4.5.3 1.4.5.4 Dok-4, and Dok-7 39 41 1.5 Gene targeting 42 1.6 Rationale and aims of this project 45 Chapter Materials and Methods 2.1 List of antibodies 47 2.2 List of primers 48 2.3 Molecular cloning methodology 48 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7 2.3.8 2.3.9 2.3.10 2.3.11 2.3.12 2.3.13 2.3.14 2.3.15 2.3.16 48 52 53 54 54 55 56 56 56 57 57 58 58 59 59 2.4 2.5 Buffers and solutions Mouse tail genomic DNA prep Genotyping of Dok-3 mice Polymerase chain reaction DNA sequencing Restriction digestion of DNA Agarose gel electrophoresis Elution of DNA from agarose gel Southern hybridization Probe labeling Dephosphorylation of plasmid DNA Ligation of DNA Preparation of DH5α competent cells Transformation of DH5α by heat shock method Bacterial DNA mini-prep by alkaline lysis Bacterial maxi-prep using Qiagen Maxi-prep columns 60 Mammalian cell culture methodology 60 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4.6 60 61 61 62 62 62 Cell culture media Purification of splenic B cells Purification of bone marrow cells Purification of thymocytes Purification of lymph node cells Proliferation assay Gene targeting methodology 63 2.5.1 Buffers and solutions 63 iii 2.5.2 2.5.3 2.5.4 2.5.5 2.5.6 2.5.7 2.5.8 2.5.9 2.5.10 2.5.11 2.6 2.7 Cloning of targeting construct Culturing of mouse embryonic fibroblasts Culturing of ES cells Preparation of DNA for transfection Transfection and selection of ES cells Picking and freezing down of ES clones in 48well plate Expanding ES clones from 48-well to 24-well plate Preparation of genomic DNA from ES cells Screening of positive ES clones Microinjection of positive ES clones 64 64 65 65 65 66 66 67 67 67 Molecular and cellular immunology methodology 68 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 Buffers and solutions Flow cytometry Enzyme-linked immuno-sorbent assay (ELISA) Preparation of Alum-precipitated antigen T-independent and T-dependent in vivo immunizations 2.6.6 Immunocytochemistry and histology 2.6.7 Intracellular calcium analysis 68 68 68 69 Protein methodology 70 2.7.1 Buffers and solutions 2.7.2 Immunoprecipitation, western blot and antibodies 2.7.3 Isolation of membrane fraction 2.7.4 Nuclear fractionation and gel shift assay 70 69 70 70 71 72 72 Chapter 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 3.3.2 Dok-3 is not required for B cell development in the peripheral immune tissues 81 84 iv 3.4 Role of Dok-3 in humoral immune responses 87 Dok-3-/- mice exhibit elevated basal serum IgM 87 Dok-3-/- mice are hyper-responsive towards Tindependent antigens 3.4.3 Loss of dok-3 does not affect T-cell dependent humoral immune responses 90 3.4.1 3.4.2 3.4.4 3.5 Aged Dok-3-/- mice did not succumb to autoimmunity Role of Dok-3 in B cell receptor activation 3.5.1 93 95 97 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 BCRstimulated Dok-3-/- B cells 101 3.5.4 Enhanced activation of JNK and p38 MAPK in 103 BCR-stimulated Dok-3-/- B cells 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 3.7.1 112 Role of Dok-3 in FcγRIIB signaling 3.7.2 Role of Dok-3 in TLR signaling 3.7.3 Interaction of Dok-3 with G3BP-1 in the immune system 113 114 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 members, including Dok-1 (p62dok), Dok-2 [p56dok-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 Nterminus Pleckstrin (PH) and Phospho-tyrosine binding (PTB) domains as well as multiple potential Src-homology (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, and 3. Dok-1 and 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 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 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 (SHIP-1), Growth factor receptorbound protein (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 Tindependent (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 hyperresponsiveness in Dok-3-/- B cells, we studied intracellular events downstream of the BCR. 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 AChR BCR BLNK BMDM Btk CML Csk DAG DNA Dok ERK ES FACS FITC FRIP G3BP G418 GANC Grb2 HA I(1,3,4,5)P4 Ig IL IP3 IRS ITAM ITIM JNK LIF LPS Lyn MAPK MAPKK MAPKKK MHC MCSF MuSK NF-κB PAMP PI(3,4)P2 PI(3,4,5)P3 PI3-K Abelson murine leukemia Acetylcholine receptor B cell receptor B cell linker protein Bone marrow-derived macrophage Bruton’s tyrosine kinase Chronic Myeloid Leukemia C-terminal Src tyrosine kinase Diacylglycerol Deoxyribonucleic acid Downstream of tyrosine kinases Extracellular-signal-regulated kinase Embryonic stem Florescence activated cell sorting Fluorescein isothiocyanate Interleukin Four Receptor Interacting Protein RasGAP SH3 domain binding protein Geneticin Gancyclovir Growth factor receptor-bound protein Haemagglutinin 1,3,4,5-tetrakisphosphates Immunoglobulin Interleukin Inositol 3,4,5-triphosphate Insulin receptor substrate Immunoreceptor tyrosine-based activation motif Immunoreceptor tyrosine-based inhibitory motif c-Jun N-terminal kinase Leukemia inhibitory factor Lipopolysaccharide Lck/yes-related novel tyrosine kinase Mitogen activated protein kinase Mitogen activated protein kinase kinase Mitogen activated protein kinase kinase kinase Major histocompatibility complex Macrophage colony-stimulating factor Muscle-specific receptor kinase Nuclear factor κB Pathogen-associated molecular pattern Phosphatidylinositol 3,4-bisphosphate Phosphatidylinositol 3,4,5-triphosphate Phosphatidylinositol 3-kinase x PI(4,5)P2 PCR PH PKB PKC PLCγ2 PTB PTEN PTK RasGAP SH2 SH3 SHIP-1 SHP-1 SOS Syk TCR TLR TNF-α Phosphatidylinositol 4,5-bisphosphate Polymerase chain reaction Pleckstrin homology Protein kinase B Protein kinase C Phospholipase C gamma Phospho-tyrosine binding Phosphatase and tensin homolog deleted on chromosome 10 Protein tyrosine kinase Ras GTPase activating protein Src-homology Src-homology SH2-containing inositol 5’-phosphatase-1 SH2-domain containing protein tyrosine phosphatase-1 Son of sevenless Spleen-associated tyrosine kinase T cell receptor Toll-like receptor Tumor necrosis factor alpha xi List of Figures Figure 1.1 mRNA expression of Dok-1, and in various mouse tissues and cell lines Figure 1.2 Expression of Dok-1, and 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 BCRstimulated 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 B cell receptor signaling pathway Diagram SHIP-1 mediated inhibition of cellular activation Diagram Structure and domains of Dok family members Diagram Amino acid sequences of chicken, mouse and human Dok-3 orthologs aligned using ClusterW algorithms Diagram Domains of Dok-3 and its binding partner Diagram T-independent and T-dependent immunization regime Diagram Structure of G3BP-1 and Table Summary of Dok family members Table Enumeration of total and lymphocytes populations in various tissues of wild-type and Dok-3 -/- mice Table 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 Introduction [...]... calcium signaling in Dok- 3- /- B cells Figure 3. 11 Enhanced NF- B activation in BCR-stimulated Dok- 3- /- B cells Figure 3. 12 Enhanced activation of JNK and p38 MAPK signaling in BCRstimulated 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... Toll-like receptor 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. .. C-terminal Src tyrosine kinase Diacylglycerol Deoxyribonucleic acid Downstream of tyrosine kinases Extracellular-signal-regulated kinase Embryonic stem Florescence activated cell sorting Fluorescein isothiocyanate Interleukin Four Receptor Interacting Protein RasGAP SH3 domain binding protein Geneticin Gancyclovir Growth factor receptorbound protein 2 Haemagglutinin 1 ,3, 4,5-tetrakisphosphates Immunoglobulin...Abbreviations Abl AChR BCR BLNK BMDM Btk CML Csk DAG DNA Dok ERK ES FACS FITC FRIP G3BP G418 GANC Grb2 HA I(1 ,3, 4,5)P4 Ig IL IP3 IRS ITAM ITIM JNK LIF LPS Lyn MAPK MAPKK MAPKKK MHC MCSF MuSK NF- B PAMP PI (3, 4)P2 PI (3, 4,5)P3 PI3-K Abelson murine leukemia Acetylcholine receptor B cell receptor B cell linker protein Bone marrow-derived macrophage Bruton’s tyrosine kinase Chronic Myeloid... Immunoglobulin Interleukin Inositol 3, 4,5-triphosphate Insulin receptor substrate Immunoreceptor tyrosine-based activation motif Immunoreceptor tyrosine-based inhibitory motif c-Jun N-terminal kinase Leukemia inhibitory factor Lipopolysaccharide Lck/yes-related novel tyrosine kinase Mitogen activated protein kinase Mitogen activated protein kinase kinase Mitogen activated protein kinase kinase kinase Major... 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... 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 B2 20+ 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... Pleckstrin homology Protein kinase B Protein kinase C Phospholipase C gamma 2 Phospho-tyrosine binding Phosphatase and tensin homolog deleted on chromosome 10 Protein tyrosine kinase Ras GTPase activating protein Src-homology 2 Src-homology 3 SH2-containing inositol 5’-phosphatase-1 SH2-domain containing protein tyrosine phosphatase-1 Son of sevenless Spleen-associated tyrosine kinase T cell receptor. .. 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... 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):1 837 -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 20 03 Aug 15;102(4): 138 1-8 xiv . 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. 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. 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

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