Mechanisms Of Akt Regulation

Akt 调节机制

• 批准号: 10394787
• 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
• 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/10394787
• 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

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.

摘要： 丝氨酸/苏氨酸蛋白激酶Akt1是参与细胞生长调控和细胞周期调控的关键信号酶。 其他生理过程，以及它的失调导致许多癌症。AKT1是一个关键的效应器 癌症促进磷脂PIP3(磷脂酰肌醇3，4，5-三磷酸)信号转导并受制于 通过C-Tail磷酸化进行调节。值得注意的是，mTORC2介导的Ser473上Akt C-Tail的磷酸化作用 一个重要的、定义明确的调控部位，通常被认为是癌症中的增殖标志。然而，如何 Ser473及辅助位点Ser477和Thr479的磷酸化调节Akt1的结构和功能 定义不明确。通过使用蛋白质半合成方法，我们的初步工作揭示了 Ser473和Ser477/Thr479上的AKT1 C-Tail磷酸化事件可以通过不同的方式缓解Akt1自身抑制 机制。我们的磷酸化Ser473激活模型表明，C端的磷酸化 AKT1可以将Pleckstrin同源(PH)结构域从激酶结构域上移走，并涉及一种磷酸 与PH-激酶连接物的相互作用。然而，PH结构域的构象动力学，连接子张力， 而磷酸-Ser477/磷酸-Thr479介导的激活的结构基础代表着关键的缺口 对Akt1规则的理解。此外，目前尚不清楚mTORC2复合体如何识别和 使Akt1的C-尾巴磷酸化，导致其激活。这项提案的目标有三个方面。1)划定 通过分析PH-激酶连接子如何通过C-尾部的磷酸化来动态调节Akt1 柔性影响pSer473的S对Akt激活的影响以及使用15N/13C/2H核磁共振谱 半合成含不同磷酸形式的节段性同位素标记Akt1的表征 Akt1的构象动力学。2)鉴定由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基于细胞的方法。3)调查结构 MTORC2复合体激活Akt1的机制基础。这些研究将提供新的基础 洞察mTORC2-Akt细胞信号轴是如何调节的，并可以为新的 抗击癌症中这些蛋白激酶调节失调的治疗策略。此外，这份职业 发展基金将增强我作为独立调查员的成功能力，通过扩大我的 实验曲目和支持额外的职业充实。也就是说，K22的支持将增强我的 沉浸在药理学、结构生物学、质谱学和基于细胞的研究中 研究环境和帮助我开始一个成功的独立学术生涯。