New insights into the regulation of Akt by its hydrophobic motif

Kearney AL, Cooke KC, Burchfield JG, Krycer JR, Fazakerley DJ and James DE

The University of Sydney, Charles Perkins Centre, Faculty of Science, School of Life and Environmental Sciences, Sydney, New South Wales, Australia.

A common hallmark amongst complex diseases such as diabetes and cancer is an aberration in cell signalling. Often implicated is the protein kinase Akt, which coordinates numerous physiological processes from cell growth to metabolism. As such, Akt is one of the most highly studied kinases, with decades of in vitro experiments dedicated to understanding its regulation, primarily by phosphorylation. For instance, Akt possesses a hydrophobic motif in its C-terminal domain that requires phosphorylation at Ser474 for it to possess kinase activity in vitro. However, we and others have new evidence that this is not the case within the cell. To demonstrate this, we generated cells ectopically expressing Akt mutants which are resistant to the Akt inhibitor MK2206. Following acute inhibition of endogenous Akt with MK2206 we analysed the ability of these mutants to facilitate insulin signalling. An Akt Ser474Ala phosphomutant facilitated the phosphorylation of several Akt substrates and enabled Akt-dependent processes such as protein synthesis and GLUT4 translocation. To dissect the mechanism by which Akt retains its activity without Ser474 phosphorylation in the cell, we performed a mutational analysis of the Akt C-terminal domain. Contrary to previous in vitroevidence, the presence of the hydrophobic motif drastically increased Akt activity even in the absence of Ser474 phosphorylation. These findings pinpoint the hydrophobic motif as a key regulator of Akt kinase activity. Furthermore, they highlight the discrepancy between the activity of kinases in vitro and in vivo, having implications for translating drug screens against kinases in diabetes and cancer research.