Mechanisms Of Akt Regulation

Akt 调节机制

基本信息

批准号：
9976789
负责人：
Nam Ky Chu
金额：
$ 18.73万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-19 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9976789
关键词：
Antineoplastic Agents Binding Biochemical Biological Assay C-terminal Cell Line Cells Complex Cryoelectron Microscopy Environment Enzymes Event FRAP1 gene Goals Grant Homologous Gene Human Immersion Isotope Labeling Knock-out Lead Length Ligation Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Measures Mediating Methods Modeling Modification Molecular Molecular Conformation NMR Spectroscopy PH Domain Pharmacology Phase Phosphatidylinositols Phospholipids Phosphorylation Phosphotransferases Physiological Physiological Processes Play Portraits Postdoctoral Fellow Process Property Protein Kinase Protein Microchips Protein NMR Spectroscopy Proteins RAC-Alpha Serine/Threonine Kinase Regulation Research Research Personnel Series Signal Pathway Signal Transduction Site Structure Tail Techniques Therapeutic Work analog base career career development cell growth regulation combat design experimental study flexibility inorganic phosphate insight novel novel strategies novel therapeutic intervention novel therapeutics platelet protein P47 structural biology tripolyphosphate tumor

项目摘要

Abstract: The Ser/Thr protein kinase Akt1 is key signaling enzyme that participates in the regulation of cell growth and other physiologic processes, and its dysregulation contributes to many cancers. Akt1 is a critical effector of the cancer promoting phospholipid PIP3 (phosphatidyl inositol 3,4,5-triphosphate) signaling and is subject to regulation by C-tail phosphorylation. Notably, mTORC2-mediated Akt C-tail phosphorylation on Ser473 acts as a major, well-defined regulatory site and is commonly measured as a proliferative mark in cancer. Yet how phosphorylation of Ser473, and auxiliary sites Ser477 and Thr479 modulate Akt1 structure and function has been poorly defined. By employing protein semisynthesis approaches, our preliminary work has revealed that Akt1 C-tail phosphorylation events at Ser473 and Ser477/Thr479 can relieve Akt1 autoinhibition using distinct mechanisms. Our model for phospho-Ser473 activation proposes that phosphorylation of the C-terminus of Akt1 can dislodge the Pleckstrin homology (PH) domain from the kinase domain and involves a phosphate interaction with the PH-kinase linker. However, the conformational dynamics of the PH domain, linker tension, and the structural basis of phospho-Ser477/phospho-Thr479-mediated activation represent key gaps in understanding of Akt1 regulation. In addition, it has been unknown how mTORC2 complex recognizes and phosphorylates the C-tail of Akt1 leading to its activation. The goals of this proposal are three-fold. 1) Delineate the dynamic regulation of Akt1 through the C-tail phosphorylation by analyzing how the PH-kinase linker flexibility influences pSer473’s impact on Akt activation and employing 15N/13C/2H NMR spectroscopy with semisynthetic segmentally isotopically labeled Akt1 containing distinct phospho forms to characterize conformational dynamics of Akt1. 2) Identify novel protein substrates phosphorylated by pSer477/pThr479 Akt1 by using protein microarrays along with other biochemical and cell-based methods. 3) Investigate the structural and mechanistic basis of Akt1 activation by mTORC2 complex. These studies will provide new fundamental insights into how the mTORC2-Akt cell signaling axis is regulated and can provide a framework for new therapeutic strategies to combat dysregulation of these protein kinases in cancer. Furthermore, this career development grant will augment my ability to succeed as an independent investigator by broadening my experimental repertoire and supporting additional career enrichment. That is, K22 support will enhance my immersion in pharmacology, structural biology, mass spectrometry, and cell-based studies in a premier research environment and help me launch a successful independent academic career.

抽象的： Ser/Thr 蛋白激酶 Akt1 是参与细胞生长调节的关键信号酶。 Akt1 参与其他生理过程，其失调会导致许多癌症。促进磷脂 PIP3（磷脂酰肌醇 3,4,5-三磷酸）信号传导的癌症，并且受到值得注意的是，mTORC2 介导的 Ser473 上的 Akt C 尾磷酸化发挥着作用。一个主要的、明确的调节位点，通常被视为癌症的增殖标志。 Ser473 的磷酸化以及辅助位点 Ser477 和 Thr479 调节 Akt1 结构和功能通过采用蛋白质半合成方法，我们的初步工作表明： Akt1 C 尾 Ser473 和 Ser477/Thr479 磷酸化事件可以使用不同的方法缓解 Akt1 自抑制我们的磷酸化 Ser473 激活模型表明，C 末端磷酸化。 Akt1 可以将 Pleckstrin 同源 (PH) 结构域从激酶结构域中移出，并涉及磷酸盐与 PH 激酶连接体的相互作用然而，PH 结构域的构象动力学、连接体张力、磷酸-Ser477/磷酸-Thr479 介导的激活的结构基础代表了此外，mTORC2 复合物如何识别和调节尚不清楚。磷酸化 Akt1 的 C 尾导致其激活该提案有三个目标 1) 描述。通过分析 PH 激酶连接子如何通过 C 尾磷酸化动态调节 Akt1 灵活性影响 pSer473 对 Akt 激活的影响，并采用 15N/13C/2H NMR 光谱半合成分段同位素标记的 Akt1，包含不同的磷酸形式来表征 Akt1 的构象动力学 2) 鉴定 pSer477/pThr479 Akt1 磷酸化的新型蛋白质底物。通过使用蛋白质微阵列以及其他生化和细胞方法 3) 研究结构。 mTORC2 复合物激活 Akt1 的机制和机制基础这些研究将为我们提供新的基础。深入了解 mTORC2-Akt 细胞信号轴如何调节，并可以为新的对抗癌症中这些蛋白激酶失调的治疗策略。发展补助金将通过扩大我的研究范围，增强我作为一名独立调查员取得成功的能力。实验曲目和支持额外的职业丰富也就是说，K22 支持将增强我的能力。沉浸在药理学、结构生物学、质谱法和基于细胞的研究中研究环境并帮助我开展成功的独立学术生涯。