Mechanisms Of Akt Regulation

摘要： 丝氨酸/苏氨酸蛋白激酶Akt 1是参与细胞生长调节的关键信号酶， 其他生理过程及其失调导致许多癌症。Akt 1是一个关键的效应器， 癌症促进磷脂PIP 3（磷脂酰肌醇3，4，5-三磷酸）信号传导，并受到 通过C尾磷酸化调节。值得注意的是，mTORC 2介导的Akt C-尾在Ser 473上的磷酸化作用， 是一个主要的、明确的调节位点，通常作为癌症的增殖标志物进行测量。但是他又怎 Ser 473的磷酸化，以及辅助位点Ser 477和Thr 479调节Akt 1的结构和功能， 定义不好。通过采用蛋白质半合成方法，我们的初步工作表明， 在Ser 473和Ser 477/Thr 479处的Akt 1 C-尾磷酸化事件可以通过使用不同的细胞因子来减轻Akt 1自身抑制。 机制等我们的磷酸化丝氨酸473激活模型提出， Akt 1可以将Pleckstrin同源（PH）结构域从激酶结构域中移出，并涉及磷酸化 与PH-激酶接头的相互作用。然而，PH结构域的构象动力学，接头张力， 磷酸化丝氨酸477/磷酸化苏氨酸479介导的激活的结构基础代表了 了解Akt 1的调控。此外，还不知道mTORC 2复合物如何识别和 磷酸化Akt 1的C-尾，导致其活化。这项建议有三个目标。1)划定 通过分析PH-激酶接头如何通过C-尾磷酸化动态调节Akt 1， 灵活性影响pSer 473对Akt活化的影响，并采用15 N/13 C/2 H NMR光谱， 含有不同磷酸形式的半合成片段同位素标记的Akt 1，以表征 Akt 1的构象动力学2)鉴定pSer 477/pThr 479 Akt 1磷酸化的新蛋白质底物 通过使用蛋白质微阵列沿着与其他生化和细胞为基础的方法。3)调查结构 和mTORC 2复合物激活Akt 1的机制基础。这些研究将提供新的基础 深入了解mTORC 2-Akt细胞信号传导轴是如何调节的，并可以为新的 治疗策略来对抗癌症中这些蛋白激酶的失调。此外，这一职业 发展补助金将扩大我的能力，以提高我作为一个独立的调查成功。 实验剧目和支持额外的职业充实。也就是说，K22支持将增强我的 沉浸在药理学，结构生物学，质谱和细胞为基础的研究在总理 研究环境，并帮助我推出一个成功的独立的学术生涯。

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.