Signaling Functions of the Tumor Suppressor CYLD

描述(申请人提供)：CyLD是一种新近发现的去泛素酶(DUB)，它负向调节由免疫受体(如肿瘤坏死因子受体(TNFR))介导的信号事件。为了了解CyLD的生理功能，我们产生了CyLD基因敲除小鼠，并进行了广泛的特性分析。这些遗传学研究揭示了细胞色素脱氢酶在调节T细胞发育和激活中的关键作用。CyLD缺陷小鼠在产生成熟胸腺细胞方面存在严重缺陷，导致外周T细胞数量减少50%以上。有趣的是，尽管CyLD-/-外周T细胞的数量较少，但它们对TCR刺激反应强烈。这些发现表明，CyLD在调节胸腺细胞发育和外周T细胞激活方面发挥着关键而独特的作用。这项修订后的应用的总体目标是了解CyLD调节T细胞发育和激活的分子机制。作为实现这一目标的关键一步，我们已将蛋白酪氨酸激酶LCK确定为细胞色素脱氢酶的特异性靶标。在TCR刺激下，CyLD与胸腺细胞中的LCK发生物理相互作用，并抑制LCK的泛素化。CyLD调节LCK与其靶标ZAP-70的可诱导结合，从而参与TCR-近端信号事件。这些发现为了解CyLD积极调节胸腺细胞发育的机制提供了重要的见解。由于CyLD负性调节外周T细胞的激活，这些发现也提出了一些有趣的问题。CyLD在调节发育中和外周T细胞的TCR信号中是否具有相反的功能？CyLD是否负向调节T细胞共刺激受体，尤其是TNFR家族成员？CyLD是作为外周T细胞的内在负性调节因子，还是通过调节胸腺细胞发育而间接起作用？另一个重要的问题是CyLD的信号功能是如何调节的。在这方面，我们已经证明了CyLD随着胸腺细胞和外周T细胞的激活而被磷酸化，从而表明了CyLD的信号功能受到其磷酸化调控的有趣的可能性。我们将执行三个具体目标，以解决这些问题并实现我们的总体目标。(1)研究CyLD调控胸腺细胞发育和TCR信号转导的分子机制。(2)研究CyLD如何负性调节外周T细胞的激活，以及CyLD缺陷是否会导致自身免疫。(3)探讨CyLD磷酸化的生化机制及其功能意义。

DESCRIPTION (provided by applicant): 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.