Elucidating structural, mechanistic, and allosteric determinants of mTOR Complex 2 (mTORC2) signaling.

马丁·泰勒|K08(PA-20-203)|项目摘要/摘要 这项提案详细说明了一项为期五年的培训计划，该计划旨在开发一项侧重于阐明 MTOR复合体2(MTORC2)信号的结构、机制和变构决定因素。磷酸化 通过mTOR复合体2(MTORC2)调控Ser473上的信号通路Akt是细胞内调控的关键步骤。 胰岛素和其他生长因子信号。Ser473磷酸化激活Akt，是Akt所必需的 下游代谢效应，如葡萄糖转运蛋白上调，这在糖尿病中是不受调节的， 以及增殖和生长，这在癌症中是不受调控的。因此，mTORC2的治疗调节 对糖尿病和癌症的治疗感兴趣，但由于两者之间的相似性，目前还不可能 MTORC2和更好地理解的效应器复合体mTORC1，它共享关键组件mTOR和 MLST8。MTORC2还激活其他底物，这些底物在离子通道稳态、凋亡、 细胞运动、转移和胰岛素受体敏感性，其活性受一系列变构调节 相互作用的蛋白质包括小的GTP酶Rho和Ras。然而，尽管mTORC2发挥着核心作用- AKT信号，关于mTORC2如何催化这一关键反应，mTORC2如何催化，我们知之甚少 识别Akt和其他底物，或这些相互作用是如何变构调节的。因此，这个项目 寻求发展对其底物的mTORC2识别的详细的结构和机械理解， 使用新的酶分析，蛋白质工程，以及生化，化学，蛋白质组学， 以及结构生物学方法，这也将开发概念验证抑制剂和潜在的激活剂 MTORC2。 我建议的研究将： (I)提供关于mTOR复合体2激酶信号的详细的结构和机制方面的见解 (2)开发适用于其他相关系统的工具、试剂和技术，包括mTORC1 (3)为我向独立的过渡提供充分的机械和翻译后续行动的机会。 我是一名执业胃肠病理学家和内科科学家，正在为有指导的研究寻求K08支持 在大卫·萨巴蒂尼博士和菲利普·科尔博士的指导下。这段80%的研究和职业生涯的辅导期 开发和20%的临床时间将确保我获得成为成功的独立人士所需的技能 首席调查员。萨巴蒂尼博士和科尔博士是国际公认的导师，共同培训了~40名 成功的独立调查人员。我的培训将在两个世界级的机构进行，怀特黑德大学 生物医学研究所和马萨诸塞州总医院病理科。两人都很富有 为青年科学家提供培训机会，追求高影响力的科学，并培养长期的 合作。我还将得到一个研究委员会的指导，他们都是各自领域的领导者：约瑟夫博士 Davis(麻省理工学院，大型蛋白质复合体结构生物学和低温电子显微镜数据分析)和施毅博士(大学 匹兹堡大学的研究成果(例如，用于大型蛋白质复合体和纳米体生成的交联质谱仪)。 这次K08奖项的支持将让我专注于成熟我的研究和加强我的事业 在指导培训的这一关键的最后阶段进行发展。在我的获奖期结束时，我将 作为一名独立的内科科学家，处于成功的最佳位置。

Taylor, Martin | K08 (PA-20-203) | PROJECT SUMMARY / ABSTRACT This proposal details a five-year training plan for the development of a research program focused on elucidating structural, mechanistic, and allosteric determinants of mTOR Complex 2 (mTORC2) signaling. Phosphorylation of the signaling kinase Akt on Ser473 by mTOR Complex 2 (mTORC2) is a critical regulated intracellular step in insulin and other growth factor signaling. Ser473 phosphorylation activates Akt and is required for Akt’s downstream metabolic effects, such as glucose transporter upregulation, which are dysregulated in diabetes, and proliferation and growth, which are dysregulated in cancer. Therapeutic modulation of mTORC2 is therefore of interest in the treatment of both diabetes and cancer but is not yet possible due to similarities between mTORC2 and the better-understood effector complex mTORC1, which shares key components mTOR and mLST8. mTORC2 also activates other substrates with less-defined roles in ion channel homeostasis, apoptosis, cell motility, metastasis, and insulin receptor sensitivity, and its activity is modulated by a series of allosteric interacting proteins including the small GTPases Rho and Ras. However, despite the central role of mTORC2- Akt signaling, we have little information about how this critical reaction is catalyzed by mTORC2, how mTORC2 recognizes Akt and other substrates, or how these interactions are modulated allosterically. This project therefore seeks to develop a detailed structural and mechanistic understanding of mTORC2 recognition of its substrates, using novel enzymologic assays, protein engineering, and a combination of biochemical, chemical, proteomic, and structural biology approaches that will also develop proof-of-concept inhibitors and potentially activators of mTORC2. My proposed studies will: (i) provide detailed structural and mechanistic insight into mTOR Complex 2 kinase signaling (ii) develop tools, reagents, and techniques applicable to other systems of interest, including mTORC1 (iii) provide ample opportunities for mechanistic and translational follow-up for my transition to independence. I am a practicing gastrointestinal pathologist and physician scientist seeking K08 support for mentored research under the guidance of Dr. David Sabatini and Dr. Philip Cole. This mentored period of 80% research and career development and 20% clinical time will ensure I acquire the skills required to become a successful independent principal investigator. Drs. Sabatini and Cole are internationally recognized mentors, together training ~40 successful independent investigators. My training will occur at two world-class institutions, the Whitehead Institute for Biomedical Research, and Massachusetts General Hospital Department of Pathology. Both are rich with opportunities for young scientists to train, pursue highly impactful science, and foster long-lasting collaborations. I will also be guided by a committee of researchers that are all leaders in their fields: Dr. Joseph Davis (MIT, structural biology of large protein complexes and cryoEM data analysis) and Dr. Yi Shi (University of Pittsburgh, crosslinking mass spectrometry as applied to large protein complexes and nanobody generation). The support of this K08 award will allow me to focus on maturing my research and strengthening my career development during this critical last stage of mentored training. At the conclusion of my award period, I will be optimally positioned for achieving success as an independent physician scientist.