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

泰勒，马丁|K08（PA-20-203）|项目总结/摘要 该提案详细介绍了一项为期五年的培训计划，旨在制定一项研究计划， mTOR复合物2（mTORC 2）信号传导的结构、机制和变构决定因素。磷酸化 通过mTOR复合物2（mTORC 2）对信号激酶Akt在Ser 473上的作用是细胞内的关键调节步骤， 胰岛素和其他生长因子信号传导。Ser 473磷酸化激活Akt，并且是Akt的 下游代谢效应，如葡萄糖转运蛋白上调，其在糖尿病中失调， 以及增殖和生长，这在癌症中失调。因此，mTORC 2的治疗性调节是 在糖尿病和癌症的治疗中感兴趣，但由于两者之间的相似性， mTORC 2和更好理解的效应复合物mTORC 1，其共享关键组分mTOR和 mLST 8。mTORC 2还激活在离子通道稳态、细胞凋亡 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GTP酶Rho和Ras。然而，尽管mTORC 2- Akt信号，我们几乎没有关于mTORC 2如何催化这个关键反应的信息，mTORC 2如何 识别Akt和其他底物，或者这些相互作用如何被变构调节。因此，该项目 试图发展mTORC 2识别其底物的详细结构和机制理解， 使用新的酶学分析、蛋白质工程以及生物化学、化学、蛋白质组学 和结构生物学方法，也将开发概念验证抑制剂和潜在的激活剂， mTORC 2。 我建议的研究将： (i)提供mTOR复合物2激酶信号传导的详细结构和机制见解 (ii)开发适用于其他相关系统（包括mTORC 1）的工具、试剂和技术 (iii)为我向独立过渡提供充分的机械和转化后续行动机会。 我是一名执业胃肠道病理学家和医生科学家，寻求K 08对指导研究的支持 在大卫萨巴蒂尼博士和菲利普科尔博士的指导下。这段时间的指导80%的研究和职业生涯 发展和20%的临床时间将确保我获得成为一个成功的独立所需的技能 首席调查员萨巴蒂尼和科尔博士是国际公认的导师，一起培训~40 成功的独立调查员我的培训将在两个世界级的机构进行，白石 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 为年轻科学家提供培训机会，追求具有高度影响力的科学， 合作。我也将由一个研究人员委员会指导，他们都是各自领域的领导者：约瑟夫博士 Davis（麻省理工学院，大型蛋白质复合物结构生物学和cryoEM数据分析）和Yi Shi博士（大学 ，如应用于大蛋白质复合物和纳米抗体生成的交联质谱法）。 这个K 08奖的支持将使我能够专注于成熟我的研究和加强我的职业生涯 在指导培训的这一关键的最后阶段进行培训。在我的获奖期结束时，我将 最适合作为一名独立的医生科学家取得成功。

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.