Mechanisms Of Akt Regulation

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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.