Regulation of TRAF2 Activity in Normal and Tumor Cells

描述(申请人提供)：TRAF2是一种关键的接头蛋白，调节IKK、JNK和p38信号通路，以响应细胞因子和压力，导致关键转录因子AP-1和NF-kB的激活。人类肿瘤经常表现出TRAF2的高表达或定位改变，导致结构性的IKK激活和对应激诱导的细胞凋亡的抵抗力增强。导致癌细胞中TRAF2稳定性和活性的这种放松调控的机制仍然不清楚。在早期的研究中，我们证明了Siah2在调节TRAF2对应激刺激的稳定性中的作用。我们还证明了TRAF2的S自身的环依赖泛素化诱导其移位到膜筏上，并伴随着JNK的激活，而不是IKK的激活。然而，调节TRAF2活性的分子机制还远远不清楚。我们定位了TNFa诱导和Akt/PKC介导的TRAF2磷酸化位点。重要的是，TRAF2环指结构域内的磷酸化是TRAF2介导的IKK激活所必需的，而TRAF结构域内的磷酸化对于限制TNFa诱导的IKK激活是重要的。这些发现为我们的假设提供了基础，即TRAF2介导的信号受到翻译后磷酸化的严格调控。在第二次修订中，我们将重点研究Akt/PKC介导的TRAF2磷酸化，并阐明其在TRAF2介导的多种信号通路激活中的生理和病理生理学意义，我们建议实现以下特定目的：1.表征新发现的TRAF2磷酸化调节TNFa诱导和TRAF2介导的JNK和NF-kB通路激活的机制。2.探讨Akt和PKC在TRAF2和TRAF2介导的JNK和NF-kB通路磷酸化中的作用。3.明确Akt和PKC介导的TRAF2磷酸化与应激诱导细胞死亡的关系。AIM-4。评估TRAF2磷酸化在黑色素瘤细胞抵抗应激诱导细胞死亡中的病理生理学相关性。我们的工作将为PKC和Akt对TRAF2磷酸化的调控以及它们在调节肿瘤细胞对应激诱导的细胞死亡的抵抗力中的作用提供新的线索。

DESCRIPTION (provided by applicant): TRAF2 is a key adaptor protein that regulates the IKK, JNK and p38 signaling pathways in response to cytokines and stress, leading to activation of the critical transcription factors AP-1 and NF-kB. Human tumors often exhibit either elevated expression or altered localization of TRAF2, resulting in constitutive IKK activation and increased resistance to stress-induced apoptosis. The mechanism that brings about such deregulation of TRAF2 stability and activity in cancer cells is still elusive. In earlier studies we demonstrated the role of Siah2 in regulating TRAF2 stability in response to stress stimulation. We also demonstrated that TRAF2's own RING-dependent ubiquitination induces its translocation to membrane rafts and concomitant activation of JNK, but not of IKK. However, the molecular mechanism underlying the regulation of TRAF2 activity is far from being understood. We mapped TNFa-induced and Akt/PKC-mediated TRAF2 phosphorylation sites. Importantly, TRAF2 phosphorylation within the RING finger domain is required for TRAF2-mediated activation of IKK, whereas phosphorylation within the TRAF domain was found important for limiting TNFa-induced IKK activation. These findings provide the foundation to our hypothesis that TRAF2-mediated signaling is tightly regulated by a post-translational phosphorylation. In this second revision, we will focus on Akt/PKC-mediated TRAF2 phosphorylation and elucidate its physiological and pathophysiological significance in TRAF2-mediated activation of diverse signaling pathways, with which we propose to carry out the following specific aims: 1. Characterize the mechanism by which newly identified TRAF2 phosphorylation regulates TNFa-induced and TRAF2-mediated activation of the JNK and NF-kB pathways. 2. Assess the role of Akt and PKC in the phosphorylation of TRAF2 and TRAF2-mediated activation of the JNK and NF-kB pathways. 3. Define the relationship between Akt- and PKC-mediated TRAF2 phosphorylation and stress-induced cell death. Aim-4. Assess the pathophysiological relevance of TRAF2 phosphorylation in melanoma cell resistance to stress-induced cell death. Our work will shed new light on the regulation of TRAF2 phosphorylation by PKC and Akt and their role in the regulation of tumor cell resistance to stress-induced cell death.