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Characterization of the Deubiquitinating Enzyme Cyld, a Novel Target of Iκb Kinase Regulating Immune Function

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Characterization of the Deubiquitinating Enzyme Cyld, a Novel Target of Iκb Kinase Regulating Immune Function The Pennsylvania State University The Graduate School Department of Cellular and Molecular Biology CHARACTERIZATION OF THE DEUBIQUITINATING ENZYME CYLD, A NOVEL TARGET OF IκB KINASE REGULATING IMMUNE FUNCTION A Thesis in Cellular and Molecular Biology By William Reiley Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2005 The thesis of William W. Reiley was reviewed and approved* by the following: Shao-Cong Sun Professor of Microbiology and Immunology Thesis Advisor Chair of Committee Robert Bonneau Associate Professor of Microbiology and Immunology Vincent Chau Professor of Cellular and Molecular Physiology Neil Christensen Associate Professor of Pathology David Spector Associate Professor of Microbiology and Immunology Henry Donahue Professor of Orthopedics and Rehabilitation Director, Department of Cell and Molecular Biology Graduate Program * Signatures are on file in the Graduate School iii ABSTRACT Since its beginnings in 1986, the transcription factor NF-κB has been implicated in the regulation of diverse biological processes ranging from development to immune responses. The pleotropic biological role of NF-κB is explained by its involvement in the regulation of growth factors, immune receptors and ligands, and apoptosis inhibitors. Knock-out mice studies have further defined the specific function of individual NF-κB members in regulating different aspects of immune function as well as the development of lymphoid and nonlymphoid tissues. In concert with functional studies, a vast amount of knowledge has been gained about how NF-κB is activated, how it is regulated and the genes which it in turn regulates. The activation of NF-κB occurs through a highly ordered pathway whereby the activation of a large kinase complex, IκB kinase (IKK), leads to the phosphorylation of IκB, its subsequent ubiquitination, proteasome-mediated degradation, and the concurrent release and nuclear translocation of NF-κB. IKK is clearly a cornerstone in the signaling pathway leading to NF-κB activation. A major missing link in the NF-κB signaling pathway is the mechanism that connects IKK to different upstream signals. It is generally believed that IKK may be activated by distinct upstream kinases in response to different stimuli. Indeed, a number of upstream kinases have been shown to activate IKK; these include members of the MAP3 kinase family, such as MEKK1, MEKK3, NIK and TAK1. Further, the physiological role of these upstream kinases in mediating receptor-specific IKK activation is becoming increasingly recognized. Connecting the MAP3Ks to the cell surface receptors, especially those belonging to the TNF receptor (TNFR) superfamily, are the adaptor proteins TRAFs. TRAFs may activate the MAP3Ks via a novel signaling iv mechanism involving ubiquitination, which occurs through lysine-63 linkages that do not target protein degradation but appear to specifically lead to signal activation. Emerging evidence suggests that TRAF-mediated ubiquitination is important for the activation of both IKK and the c-Jun N-terminal kinase (JNK) by various TNFR members, as well as certain other immune receptors, such as the toll-like receptors and IL-1 receptors. It is thus apparent that the ubiquitination activity of TRAFs must be under tight control. Recent biochemical studies have identified a negative regulator of TRAF- ubiquitination. This factor, CYLD, was originally discovered as a tumor suppressor that is mutated in cylindromatosis, a predisposition to tumors of skin appendages. CYLD belongs to the deubiquitination enzyme (DUB) family, which digests ubiquitin chains. At least in transfected cells, CYLD inhibited the ubiquitination of two major TRAF members, TRAF2 and TRAF6, as well as the ubiquitination of a downstream target, IKKγ. Consistent with its DUB activity, CYLD negatively regulates the activation of NF-κB by TRAFs and TNFRs. These in vitro studies provide an insight into the tumor suppressor function of CYLD and shed light on the signaling role of this DUB. However, the physiological function of CYLD in the immune system and other biological systems remain unclear. Hence, the work described in this thesis was aimed at understanding the physiological role of CYLD. A number of unique findings are presented here: 1) CYLD is a non-redundant DUB of TRAF2. Although initial reports suggest that overexpressed CYLD inhibits the ubiquitination of transfected TRAF2, it has remained unclear whether CYLD indeed serves as a critical regulator of TRAF2 under endogenous conditions. By RNA interference-(RNAi)- v mediated CYLD knock down, we demonstrated that the loss of endogenous CYLD resulted in constitutive ubiquitination of endogenous TRAF2. This finding suggest that CYLD is a key DUB that controls the state of TRAF2 ubiquitination. 2) CYLD is a negative regulator of both IKK and JNK. Our CYLD knock down studies reveal that the loss of endogenous CYLD caused hyperactivation of not only IKK, but also JNK. Further, the ability of CYLD to regulate JNK was specific for immune receptors and involved the upstream kinase MKK7, but not MKK4. 3) The DUB function of CYLD is negatively regulated by its phosphorylation. CYLD becomes transiently phosphorylated along with signal-induced activation of JNK and IKK. We mapped the phosphorylation sites of CYLD to seven serines. Interestingly, the phosphorylation of CYLD appears to involve IKK, and this posttranslational modification caused inactivation of CYLDs ability to deubiquitinate TRAF2. Consequently, CYLD mutants that are defective in phosphorylation prevents signal-induced TRAF2 ubiquitination and a loss in the negative regulation of JNK and IKK. These findings suggest a model where CYLD phosphorylation serves as a mechanism to inactivate its DUB function, thus allowing transient activation of TRAF2 ubiquitination and initiation of downstream signaling pathways. 4) Establishment of CYLD as an immune regulator. In order to understand the physiological functions of CYLD, we generated CYLD knockout mice. Although these mutant mice had no gross abnormalities in growth or survival, vi they displayed clear defects in the immune system. First, the number of peripheral T cells was significantly reduced in the spleens of CYLD -/- mice. This defect was at least partially due to a defect in thymocyte development from the double-positive to the single-positive stage, which was more pronounced for CD4+ T cells. Second, CYLD -/- T cells were hyper- responsive to TCR stimulation, producing markedly larger amounts of cytokines than control T cells. These findings suggest that CYLD is a negative regulator of T cell activation, where it is required for T cell development in the thymus. Since it is the step of negative selection that appears to be affected by the loss of CYLD, the CYLD deficiency may cause abnormal TCR signaling, resulting in uncontrolled T cell deletion during this stage of development. In summary, the work presented here 1) defines a signaling function for CYLD in regulating TRAF ubiquitination and activation of NF-κB and JNK, 2) elucidates a mechanism of CYLD regulation, which involves its site specific phosphorylation and 3) demonstrates a role for CYLD in the regulation of T cell development and activation. These results provide significant insight into the physiological function of CYLD as well as the mechanism of its regulation. These findings also provide an example for how a DUB participates in signal regulation of the immune system. vii TABLE OF CONTENTS Page LIST OF FIGURES………………………………………………………………….. ix LIST OF ABBREVIATIONS………………………………………………………...x ACKNOWLEDGMENTS…………………………………………………………….xiii CHAPTER I. LITERATURE REVIEW……………………………………………..1 1.1 The Two Branches of the Immune System…………………. 2 1.2 NF-κB Signal Transduction Pathway Regulates Both the Innate and Adaptive Immune Functions………….. 5 1.3 NF-κB Family and Structure………………………………...5 1.4 Inhibitors of κB………………………………………….….. 8 1.5 Mechanism of NF-κB Activation……………………………10 1.6 The IκB kinase (IKK)………………………………………..14 1.7 Receptors Mediating Activation of IKK and NF-κB……….. 22 1.7.1 Toll-like Receptors…….……………………22 1.7.2 Antigen Receptors……...…………………...29 1.7.3 Tumor Necrosis Factor Receptor Superfamily……………………… 36 1.8 Tumor Necrosis Factor Receptor Associated Factors (TRAFs)…………………………….….. 40 1.9 Role of TRAF2s in Activation of JNK and IKK…………….42 1.10 The Role of TRAF6 in Activation of JNK and IKK………... 43 1.11 Ubiquitination and Deubiquitination……………….………..45 1.12 CYLD a Novel DUB Involved in Regulation of of Signal Transduction……………………………………… 47 CHAPTER II. NEGATIVE REGULATION OF JNK SIGNALING BY THE TUMOR SUPPRESSOR CYLD………………………..,.. 52 Abstract……………………………………………………………... 53 Introduction…………………………………………………………. 54 Materials and Methods……………………………………………… 56 Results………………………………………………………………. 58 Discussion…………………...……………………………………… 63 Acknowledgments………………………………………………...… 66 CHAPTER III. REGULATION OF THE DEUBIQUITINATING ENZYME CYLD BY IKKγ-DEPENDENT PHOSPHORYLATION …………………...………………………..76 Abstract………………………………………………………………77 Introduction……………………………………………………….… 78 Materials and Methods……………………………………………… 80 Results………………………………………………………………. 84 viii Discussion…………………………………………………………... 93 Acknowledgments…………………………………………………... 97 CHAPTER IV. REGULATION OF T CELL DEVELOPMENT AND ACTIVATION BY THE DEUBIQUITINATION ENZYME CYLD………………………….……………………….. 116 Abstract……………………………………………………………... 117 Introduction……………………………………………………….… 118 Materials and Methods……………………………………………… 121 Results………………………………………………………………. 124 Discussion…………………………………………………………..
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