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

定义 TSC 肿瘤抑制因子在 mTORC1 信号转导中的生化功能

• 批准号: 10392665
• 负责人: Sophie Morgan Evarts
• 金额: $ 0.25万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2021-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392665
• 关键词: Address; Affect; Binding; Biochemical; Biological Assay; Cell Line; Cells; Complex; Data; Epitopes; Event; Experimental Designs; FRAP1 gene; GTP Binding; GTPase-Activating Proteins; Genetic; Growth; Growth Factor; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate; Hela Cells; Human; Immunofluorescence Microscopy; In Vitro; Lead; Length; Lung diseases; Lymphangioleiomyomatosis; Malignant Neoplasms; Maps; Measures; Methods; Missense Mutation; Molecular; Monomeric GTP-Binding Proteins; Mutation; Nucleotides; Oncogenic; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Proteins; Protocols documentation; Public Health Schools; Publishing; Ras homolog enriched in brain; Reagent; Regulation; Research; Serum; Signal Pathway; Signal Transduction; Stimulus; Surface; Syndrome; TSC1 gene; TSC2 gene; Techniques; Tuberous sclerosis protein complex; Tumor Suppressor Genes; Tumor Suppressor Proteins; base; cell growth; crosslink; experimental study; insight; loss of function; loss of function mutation; mutant; neoplastic; novel; protein complex; tumor; tumor growth; uncontrolled cell growth; upstream kinase

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.

项目摘要/摘要 许多生长因子信号通路在结节硬化症复合物（TSC）复合物上收敛以激活 雷帕霉素复合物1（MTORC1）的机械目标，这是合成代谢过程促进的关键事件 潜在的细胞生长。 TSC复合物由肿瘤抑制剂TSC1和TSC2组成，以及 蛋白质TBC1D7。在复合物中，TSC2充当GTPase激活蛋白（GAP）朝向小的G- 蛋白质Rheb，当它与GTP结合时会有力激活MTORC1。控制mtorc1的途径 由一些人类中的一些最常见的癌基因和肿瘤抑制剂组成 癌症。此外，在TSC1和TSC2本身中发现的突变已在零星癌中鉴定出来。 在这些情况下，MTORC1被异常激活，导致细胞生长不受控制。虽然很重要 在理解MTORC1信号网络方面取得了进展，这是许多长期存在的问题 TSC复合物及其对RHEB的调节的基本机械功能仍未得到解答。我们的 实验室最近确定了通过生长因子信号激酶在TSC2上的特定磷酸化事件， AKT，通过TSC复合物的空间定位来调节MTORC1活性。但是，更深了 对磷酸化如何影响TSC2-RHEB结合和RHEB-GDP/GTP状态的机械理解， 以及是否需要其他激酶调节复合物的定位。此外，TSC2如何与 不同核苷酸结合状态的RHEB以及TSC复合物是否包含调节功能 独立于间隙活性的Rheb尚不清楚。该建议将解决这些未解决的问题 关于通过生化研究的TSC复合调节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信号传导实验。总的来说，这些机制 研究将为TSC肿瘤抑制子的调节和功能提供新的分子见解和 它们在正常情况和癌症中对Rheb和MTORC1的调节。为了调查这些问题，我 提出以下目的：1）映射TSC2-RHEB结合域并表征新的调节 这些区域固有的功能。 2）确定上游激酶如何通过上游激酶对TSC2的多位磷酸化 影响TSC2-RHEB结合和调节。该提案中概述的研究将在 哈佛T.H. Brendan Manning博士实验室的Chan公共卫生学院。曼宁博士是 MTORC1领域，在生化技术和实验设计方面具有广泛的专业知识。此外， 曼宁实验室已经建立了许多建议的试剂和协议。