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

Abstract

Edited by Alex TokerThe 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 ϳ50% 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.

Silencing branched-chain ketoacid dehydrogenase or treatment with branched-chain ketoacids ex vivo inhibits muscle insulin signaling

“…Interestingly, changes in BCKDHA content profoundly affected AKT2 Ser 474 phosphorylation than on AKT1 Ser 473. Previous studies have reported the importance of AKT2 Ser 474 phosphorylation for maximal activation of AKT and insulin-regulated processes (82).…”

Control of Akt activity and substrate phosphorylation in cells

“…Using a compartment‐specific probe for the endogenous mTORC2 activity, it was shown that both the localization and activity of mTORC2 at the plasma membrane are PI3K‐independent and that phosphorylation of Akt in response to growth factors is exclusively triggered by its membrane recruitment . However, it is important to keep in mind that mTORC2 is not absolutely required for Akt activity, though it augments phosphorylation of several Akt‐specific substrates, such as FoxO transcription factors, Tsc2, PRAS40, and AS160 …”

“…Similarly, phosphorylation of the lysosomal Akt substrate GFAP was shown to inhibit chaperone‐mediated autophagy . Furthermore, Akt phosphorylation on S473 could differentially regulate glucose transporters Glut1 and Glut4 in different cell types, suggesting that compartment‐specific phosphorylation dynamics could help define functional outcomes of Akt signaling. These examples demonstrate that cellular membranes could both contribute to local Akt activation kinetics and guide its activity toward compartment‐specific substrates.…”

“…However, recent studies using protein semisynthesis and genetic code expansion demonstrated that while phosphorylation of S473 increased activity of T308‐phosphorylated Akt in vitro , the former site is essentially dispensable for the activity in cells . Indeed, mutation of S473 or genetic ablation of mTORC2 components have only minor effect on cellular Akt phosphorylation and activity …”

“…Thus, phosphorylation of S473 was proposed to help protect Akt from dephosphorylation by cellular phosphatases, although to which extent this is due to the PIF pocket occupancy or Akt membrane binding (discussed in the following) is currently unclear. It is also possible that engagement of the PIF pocket could help stabilize the active conformation of the kinase and/or modulate Akt substrate specificity in cells; future studies should help gauge contribution of the HM‐dependent mechanisms to cellular Akt activity.…”