Defining the Biochemical Functions of the TSC Tumor Suppressors in mTORC1 Signaling

项目总结/摘要 许多生长因子信号通路汇聚在结节性硬化症复合体（TSC）上， 雷帕霉素复合物1（mTORC 1）的机制靶点，促进合成代谢过程的关键事件 潜在的细胞生长。TSC复合物由肿瘤抑制因子TSC 1和TSC 2以及肿瘤抑制因子TSC 1和TSC 2组成。 TBC 1D 7蛋白。在复合物中，TSC 2作为GT3活化蛋白（GAP）作用于小G- Rheb蛋白，当mTORC 1与GTP结合时，其可有效激活mTORC 1。控制mTORC 1的途径 是由一些最常见的致癌基因和肿瘤抑制基因在人类中发生遗传改变组成的 癌的此外，已经在散发性癌症中鉴定了在TSC 1和TSC 2本身中发现的突变。 在这些情况下，mTORC 1被异常激活，导致细胞生长不受控制。虽然显著 在理解mTORC 1信号网络方面取得了进展，许多长期存在的问题 TSC复合物的机械功能及其对Rheb的调节的潜在机制仍然没有答案。我们 实验室最近通过生长因子信号激酶确定了TSC 2上特异性磷酸化事件， Akt通过TSC复合物的空间定位调节mTORC 1活性。然而，更深层次的 磷酸化如何影响TSC 2-Rheb结合和Rheb-GDP/GTP状态的机制理解， 以及是否需要另外的激酶调节复合物的定位。此外，TSC 2如何与 不同核苷酸结合状态下的Rheb以及TSC复合物是否包含对 不依赖于GAP活性的Rheb是未知的。这项提案将解决这些悬而未决的问题 通过生物化学研究，了解TSC复合体调节和功能的分子决定因素 包括体外TSC 2-Rheb结合、Rheb核苷酸交换和TSC 2激酶测定，以及细胞免疫测定。 基于免疫荧光显微镜和mTORC 1信号转导实验。总的来说，这些机械 研究将为TSC肿瘤抑制因子的调节和功能提供新的分子见解， 它们在正常环境和癌症中对Rheb和mTORC 1的调节。为了研究这些问题，我 提出以下目的：1）定位TSC 2-Rheb结合结构域并表征新的调控结构域 这些区域的固有功能。2)确定上游激酶如何多位点磷酸化TSC 2 影响TSC 2-Rheb结合和调节。本提案中概述的研究将在 哈佛TH陈公共卫生学院在布伦丹曼宁博士的实验室。曼宁博士是 mTORC 1领域，并在生化技术和实验设计方面拥有丰富的专业知识。此外，本发明还 许多所提出的试剂和方案已经在曼宁实验室中建立。

Project Summary/Abstract Many growth factor signaling pathways converge on the tuberous sclerosis complex (TSC) complex to activate the mechanistic target of rapamycin complex 1 (mTORC1), a key event in the promotion of anabolic processes underlying cell growth. The TSC complex is composed of the tumor suppressors, TSC1 and TSC2, and the protein TBC1D7. Within the complex, TSC2 acts as a GTPase activating protein (GAP) towards the small G- protein Rheb, which potently activates mTORC1 when it is GTP bound. The pathways that control mTORC1 are comprised of some of the most common oncogenes and tumor suppressors genetically altered in human cancers. Moreover, mutations found in TSC1 and TSC2 themselves, have been identified in sporadic cancers. In these settings, mTORC1 is aberrantly activated and results in uncontrolled cell growth. Although significant progress has been made in understanding the mTORC1 signaling network, many longstanding questions underlying the mechanistic function of the TSC complex and its regulation of Rheb remain unanswered. Our lab recently established that specific phosphorylation events on TSC2 by the growth factor signaling kinase, Akt, regulates mTORC1 activity through spatial localization of the TSC complex. However, a deeper mechanistic understanding of how phosphorylation impacts TSC2-Rheb binding and the Rheb-GDP/GTP state, and whether additional kinases regulate localization of the complex is needed. Moreover, how TSC2 binds to Rheb in different nucleotide-bound states and whether the TSC complex contains regulatory functions towards Rheb independent of GAP activity are unknown. This proposal will address these unanswered questions regarding the molecular determinants of TSC complex regulation and function through biochemical studies including in vitro TSC2-Rheb binding, Rheb nucleotide exchange and TSC2 kinase assays as well as cell based immunofluorescence microscopy and mTORC1 signaling experiments. Collectively, these mechanistic studies will provide novel molecular insights into the regulation and function of the TSC tumor suppressors and their regulation of Rheb and mTORC1 in both normal settings and cancer. To investigate these questions, I propose the following aims: 1) Map the TSC2-Rheb binding domains and characterize novel regulatory functions inherent to these regions. 2) Determine how multi-site phosphorylation of TSC2 by upstream kinases influences TSC2-Rheb binding and regulation. The research outlined in this proposal will take place at the Harvard T.H. Chan school of Public Health in the lab of Dr. Brendan Manning. Dr. Manning is an expert in the mTORC1 field and has extensive expertise in biochemical techniques and experimental design. Additionally, many of the proposed reagents and protocols have already been established in the Manning lab.