Mechanistic Understanding of PH Domain-Mediated Autoinhibition and Oncogenic Activation of Akt

Abstract

The protein kinase Akt plays a critical role in cell survival and proliferation, and its over-activation can drive cancer. The activity of Akt is delicately regulated by post-translational modifications (PTMs) and its regulatory domains. In particular, phosphorylation on Akt’s activation loop and regulatory C-tail has been demonstrated to be important in stimulating its kinase activity. It is known that the N-terminal pleckstrin homology (PH) domain of Akt plays a key role in autoinhibiting Akt and preventing unregulated pro-growth signaling via an intramolecular interaction with the kinase domain. However, the precise PH domain-mediated Akt autoinhibition mechanisms remain unclear. In this study, we have unexpectedly found that Ala mutation at Arg86 (R86A) strengthens the inhibitory role of the PH domain by promoting the interdomain interaction between the PH and kinase domains of Akt regardless of its C-tail phosphorylation status. Our structural and biophysical analysis of the R86A mutant has led to the characterization of two key PH domain residues, Glu17 and Tyr18 that control Akt autoinhibition. The hydrogen bond network involving Arg86, Tyr18, and Glu17 governs the catalytic activity of Akt and influences the rate of activation loop dephosphorylation by protein phosphatase PP2A. Moreover, our studies show that a well-established oncogenic form of Akt, E17K, displays elevated Akt kinase activity of the non-C-tail phosphorylated form but the unchanged affinity for soluble phosphatidylinositol (3,4,5)-trisphosphate (PIP3). These findings contrast a commonly accepted model previously reported for the oncogenic mechanisms of E17K. Through in-depth biochemical, cellular, and structural studies assisted by the Google AlphaFold, we have developed a model to explain how Glu17 and Tyr18 on the PH domain play a critical role to autoinhibit the enzymatic activity of Akt. These results provide a new framework for understanding how oncogenic mutations at the interface between the PH and kinase domains, such as E17K, activate Akt and suggest a mechanism by which the PH domain governs Akt autoinhibition to control Akt signaling.