CYLD is a recently identified deubiquitinating enzyme (DUB) that negatively regulates the signaling events mediated by immune receptors, such as TNF receptors (TNFRs). To understand the physiological function of CYLD, we have generated CYLD knockout mice and undertaken extensive characterization analyses. These genetic studies have revealed a critical role for CYLD in regulating T-cell development and activation. CYLD-deficient mice have a severe defect in generating CD4 and CD8 mature thymocytes, resulting in more than 50% reduction in peripheral T-cell numbers. Interestingly, despite their lower numbers, the CYLD-/- peripheral T cells are hyper-responsive to TCR stimulation. These findings suggest that CYLD plays critical but distinct roles in regulating thymocyte development and peripheral T-cell activation. The overall objective of this revised application is to understand the molecular mechanism by which CYLD regulates T-cell development and activation. As a critical step towards achieving this objective, we have identified the protein tyrosine kinase LCK as a specific target of CYLD. CYLD physically interacts with LCK in thymocytes in response to TCR stimulation and inhibits the ubiquitination of LCK. CYLD regulates the inducible binding of LCK to its target ZAP-70, thereby participating in TCR-proximal signaling events. These findings provide an important insight into the mechanism by which CYLD positively regulates thymocyte development. Since CYLD negatively regulates peripheral T-cell activation, these findings also raise a number of intriguing questions. Does CYLD possess opposing functions in regulating TCR signaling of developing and peripheral T cells? Does CYLD negatively regulate T-cell costimulatory receptors, especially TNFR family members? Does CYLD serve as an intrinsic negative regulator of peripheral T cells or act indirectly through regulating thymocyte development? Another important question is how the signaling function of CYLD is regulated. In this regard, we have shown that CYLD is phosphorylated along with the activation of both thymocytes and peripheral T cells, thus suggesting the intriguing possibility that the signaling function of CYLD is subject to regulation by its phosphorylation. We will perform three specific aims to address these questions and to achieve our overall objective. (1) Characterize the molecular mechanism by which CYLD regulates thymocyte development and TCR signaling. (2) Examine how CYLD negatively regulates peripheral T-cell activation and whether the CYLD deficiency causes autoimmunity. (3) Investigate the biochemical mechanism and functional significance of CYLD phosphorylation.