IDENTIFICATION OF CYCLIN-DEPENDENT KINASE 5 IN T CELLS AND ITS ROLE IN REGULATING T CELL FUNCTION AND DIFFERENTIATION By ERIC M. LAM Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. John J. Letterio, MD Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ERIC M LAM candidate for the degree of Doctor of Philosophy degree *. Committee Members Noa Noy (Committee Chair) John J Letterio (Mentor) Bing-Cheng Wang Anthony Berdis Marvin Nieman Date of Defense January 13, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication To my parents, who scarified everything for their children to have a better life, and taught us to work hard, work smart, and to love your family and friends. To my sister who has always inspired me and taken care of me. Thank you. Table of Contents Table of Contents i List of Figures/Tables iv Acknowledgements vi Abstract 1 Chapter 1: Introduction 3 1.1 Overview of Cyclin-dependent kinase 5 (Cdk5) 4 1.1.1 History of Cdk5 discovery and introduction to an atypical CDK 4 1.1.2 Regulation of Cdk5 activity 5 1.1.3 Function of Cdk5 in the nervous system 6 1.1.4 Dysregulation of Cdk5 in disease and disorder 9 1.1.5 Cdk5 and its role in non-neuronal tissues 10 1.2 The Immune System and Regulatory T cells 13 1.2.1 T cell subsets 14 1.2.2 The role of IL2 in T cell function and the regulation of IL2 15 gene transcription 1.2.3 Regulatory T cells and their role in immune homeostasis 18 and disease 1.2.4 Foxp3+ expression and function in regulatory T cells 20 1.2.5 Regulation of Foxp3 expression 22 1.3 Statement of Purpose 25 Chapter 2: Cyclin-dependent kinase 5 activity is required for T cell activation and induction of experimental autoimmune encephalomyelitis 35 2.1 Abstract 35 i 2.2 Introduction 36 2.3 Materials and Methods 39 2.4 Results 47 2.5 Discussion 55 Chapter 3: Cdk5 controls IL-2 Gene Expression via Repression of 85 the mSin3a-HDAC Complex 3.1 Abstract 85 3.2 Introduction 86 3.3 Materials and Methods 87 3.4 Results 91 3.5 Discussion 96 Chapter 4: Cyclin-dependent kinase 5 represses Foxp3 gene expression and Treg development through specific phosphorylation of Stat3 at Serine 727 113 4.1 Abstract 113 4.2 Introduction 114 4.3 Materials and Methods 117 4.4 Results 120 4.5 Discussion 125 Chapter 5: Summary, Conclusions, Discussion and Future Directions 138 5.1 Summary 138 5.2 Conclusions: 139 5.2.1 Chapter 2: Cdk5 activity is required for T cell activation and induction of experimental autoimmune encephalomyelitis 139 5.2.2 Chapter 3: Cdk5 controls IL-2 Gene Expression via Repression of the mSin3a-HDAC Complex 141 ii 5.2.3 Chapter 4: Cdk5 regulates Foxp3 expression in CD4+ T cells through modification of Stat3 phosphorylation 143 5.3 Discussion and Future Directions 144 5.3.1 Chapter 2 – Discussion and Future Directions 144 5.3.2 Chapter 3 – Discussion and Future Directions 151 5.3.3 Chapter 4 – Discussion and Future Directions 154 References 165 iii List of Figures/Tables Figure 1.1 Cdk5 activity and the Cdk5 substrate consensus sequence 27 Figure 1.2 The murine cerebral cortex is characterized by six distinct layers of neurons 29 Figure 1.3 Mechanisms of suppression used by Treg cells 31 Figure 2.1 Increased Cdk5 activity in the CNS of mice with EAE 61 Figure 2.2 Cdk5 activity is up-regulated in lymphocytes after TCR or antigen specific stimulation 63 Figure 2.3 Disruption of Cdk5 activity abrogates antigen receptor Mediated lymphocyte activation 65 Figure 2.4 Abrogation of Cdk5 activity in lymphocytes ameliorates clinical signs and associates with reduced pathology in EAE 67 Figure 2.5 Cdk5 physically interacts with and phosphorylates coronin 1a 69 Figure 2.6 Disruption of Cdk5 activity diminishes actin polarization and migration towards CCL19 71 Figure 2.7 Inhibition of Cdk5 activity by roscovitine treatment abrogates antigen receptor mediated lymphocyte activation 73 Figure 2.8 Generation and characterization of immune chimeric mice and their immunophenotyping 75 Figure 2.9 Characterization of immune cell infilterate in spinal cord after EAE induction 77 Figure 2.10 Arp2/3 failed to polarize in Cdk5-/-C encephalitogenic lymphocytes 79 Figure 2.11 Roscovitine treatment disrupts migration of murine T cells and human Jurkat cells towards CCL19 in a transmigration assay 81 Figure 2.12 In vitro cytokine production by encephalitogenic splenocytes following restimulation with MOG (35-55) 83 Figure 3.1 Cdk5 is required for optimal IL-2 expression 103 iv Figure 3.2 Cdk5 interacts with HDAC1 and alters HDAC activity in activated T cells 105 Figure 3.3 Cdk5 does not phosphorylate HDAC1 but does phosphorylate the mSin3a protein at serine residue 861 107 Figure 3.4 Cdk5 phosphorlyation of mSin3a at Serine861 disrupts the expression of the mSin3a protein 109 Figure 3.5 Phosphorylation of mSin3a by Cdk5 disrupts the formation of the HDAC1/mSin3a complex 111 Figure 4.1 Cdk5 is essential for IL6 suppression of TGF-β induced Foxp3 expression in CD4+ T cells 130 Figure 4.2 Cdk5 activity does not alter Smad activation or binding to the Foxp3 gene 132 Figure 4.3 Cdk5 physically interacts with and phosphorylates Stat3 at Serine 727 134 Figure 4.4 Cdk5 is required to induce Stat3 binding to the Foxp3 gene 136 Figure 5.1 Increased Cdk5 expression in ALL patient samples 159 Figure 5.2 Increased Stat3 phosphorylation detected in ALL patient samples 161 Table 1.1 Non-neuronal substrates of Cdk5 33 v Acknowledgments I have several people to thank for guiding me along this path towards my PhD. Thank you first and foremost to my mentor Dr. John Letterio for providing support, guidance and encouragement throughout my training. You have always been a positive force and your vast knowledge and love for science inspires me to keep searching for the truth. Thank you to my committee members, Dr. Noa Noy, Dr. Anthony Berdis, Dr. Bing- Cheng Wang and Dr. Marvin Nieman. Thank you for the interesting, insightful and at times tricky questions. You kept me thinking and pushed my science. Thank you to Dr. Tej Pareek, Dr. Byung-Gyu Kim and Dr. Sung Hee Choi for all your help in the laboratory. You have taught me so many things both at the bench and in life. Special thanks to my friends and all those close to my heart. Each and everyone of you have made an impact on me during this journey. You have pulled me through the bad times and rode with me during the good. Thanks for always believing in me and being there when I was in need. Thank you to my sister Josephine, and my brother-in-law, Ricky. The two of you have been an inspiration of how to balance being successful at work and loving your family. Thank you for always being there for me when I needed you guys and for being the lead actors in my real-life “Friends” episode. Finally, and most importantly, thank you to my parents for always believing in me and supporting me with your constant encouragement. You have shaped me into the person I am today and I could not have done gotten to this point without your love. vi Identification of Cyclin-dependent kinase 5 in T cells and its Role in Regulating T Cell Function and Differentiation Abstract By ERIC LAM Cdk5 is a unique member of a family of serine/threonine cyclin-dependent kinases. Although Cdk5 is ubiquitously expressed, this kinase is predominantly active in post- mitotic neurons where there is abundant expression of its obligate activating partners p35 and/or p39. Consequently, Cdk5 has been historically considered a ‘neuron-specific’ kinase and recognized as an essential regulator of neuronal functions. However, aberrant Cdk5 activity has be associated with a variety of inflammatory neurodegenerative disorders and activity of the Cdk5-p35 complex has been associated with disorders involving non-neuronal tissues, indicating the potential for novel extra-neuronal roles for Cdk5. Our work describes a previously unknown function of the Cdk5-p35 complex in T cells. We demonstrate the activity of Cdk5 is required for the induction of EAE, a T-cell- mediated neuro-inflammatory disease model of multiple sclerosis. Activation of T cells leads to an induction of Cdk5-p35 expression that we show is essential for optimal T cell function. In an effort to define mechanisms mediating this effect of Cdk5, we discovered that Cdk5 controls IL2 gene expression during T-cell activation by modulating the HDAC1/mSin3a repressor complex. We show this effect of Cdk5 is dependent on specific phosphorylation of the mSin3a protein at serine 861. Disruption of Cdk5 activity 1 in T cells results in enhanced HDAC activity and binding of the HDAC1/mSin3a repressor complex to the IL2 promoter and subsequently leads to the suppression of IL2 expression. In our pursuit of alternative mechanisms, we focused on Foxp3+ regulatory T cells (Tregs) whose activity is essential for maintaining immune homeostasis and controlling inflammation in the CNS. We show that Cdk5 activity is a determinant of the expression of Foxp3 in CD4+ T cells, effectively regulating the differentiation of Tregs in the presence of pro-inflammatory factors. Our data show the IL-6 induced repression of Foxp3 gene expression requires Cdk5-mediated phosphorylation of Stat3 specifically at residue 727.