Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 294, Issue 45, Pages 16729-16739Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.010036
Keywords
Akt PKB; serine; threonine protein kinase; phosphorylation; cell signaling; adipocyte; insulin; substrate specificity; mTOR complex (mTORC); protein synthesis; glucose transport; Akt Ser(474) phosphorylation; Akt W80A; chemical genetics; GLUT4; MK2206
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Funding
- Australian Research Council [DP180103482]
- National Health and Medical Research Council [GNT1120201]
- Diabetes Australia [Y19G-FAZD]
- Chen Family research scholarship
- National Health and Medical Research Council
- Australian Diabetes Society Skip Martin early career fellowship
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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.
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