Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser ⁴⁷⁴ ; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser ⁴⁷⁴ phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser ⁴⁷⁴ phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser ⁴⁷⁴ phosphorylation in 3T3-L1 adipocytes by preventing Ser ⁴⁷⁴ phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser ⁴⁷⁴ phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser ⁴⁷⁴ phosphorylation. We propose a model where Ser ⁴⁷⁴ phosphorylation is required for maximal Akt2 kinase activity in adipocytes.
(© 2019 Kearney et al.)