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

项目摘要/摘要 许多生长因子信号通路聚集在结节性硬化症复合体(TSC)上激活 促合成代谢过程中的关键事件雷帕霉素复合体1(MTORC1)的作用靶点 潜在的细胞生长。TSC复合体由肿瘤抑制因子TSC1和TSC2以及 蛋白TBC1D7。在复合体中，TSC2作为GTP酶激活蛋白(GAP)朝向小G- 蛋白Rheb，当mTORC1与GTP结合时，它会有效地激活mTORC1。控制mTORC1的通路 由一些人类最常见的癌基因和肿瘤抑制基因组成 癌症。此外，在TSC1和TSC2本身中发现的突变已经在散发性癌症中被发现。 在这些情况下，mTORC1被异常激活，导致细胞生长失控。虽然意义重大 在理解mTORC1信令网络方面取得了进展，许多长期存在的问题 TSC复合体的机制及其对Rheb的调控仍然没有答案。我们的 实验室最近发现，TSC2上的特异性磷酸化事件是通过生长因子信号转导激酶发生的， AKT，通过TSC复合体的空间定位来调节mTORC1的活性。然而，更深一层的 从机制上理解磷酸化如何影响TSC2-Rheb结合和Rheb-GDP/GTP状态， 以及是否需要额外的激酶来调节复合体的定位。此外，TSC2如何绑定到 Rheb在不同的核苷酸结合状态以及TSC复合体是否包含调节功能 Rheb不依赖于GAP活性是未知的。这项提案将解决这些悬而未决的问题 通过生化研究探讨TSC复合体调控和功能的分子决定因素 包括体外TSC2-Rheb结合、Rheb核苷酸交换和TSC2激酶测定以及细胞 基于免疫荧光显微镜和mTORC1信号转导实验。总而言之，这些机械论 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 他们在正常情况下和癌症中对Rheb和mTORC1的调节。为了调查这些问题，我 提出了以下目标：1)定位TSC2-Rheb结合域并鉴定新的调控结构域 这些地区固有的功能。2)确定上游激酶对TSC2的多位点磷酸化 影响TSC2-Rheb的结合和调节。本提案中概述的研究将在 哈佛大学公共卫生学院，布伦丹·曼宁博士的实验室。曼宁博士是这方面的专家 他在生化技术和实验设计方面拥有丰富的专业知识。另外， 许多拟议的试剂和方案已经在曼宁实验室建立。

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.