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

Abstract

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 Ser⁴⁷⁴; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser⁴⁷⁴ phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser⁴⁷⁴ phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser⁴⁷⁴ phosphorylation in 3T3-L1 adipocytes by preventing Ser⁴⁷⁴ 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 Ser⁴⁷⁴ phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser⁴⁷⁴ phosphorylation. We propose a model where Ser⁴⁷⁴ phosphorylation is required for maximal Akt2 kinase activity in adipocytes.

Keywords: Akt PKB; Akt Ser474 phosphorylation; Akt W80A; GLUT4; MK2206; adipocyte; cell signaling; chemical genetics; glucose transport; insulin; mTOR complex (mTORC); phosphorylation; protein synthesis; serine/threonine protein kinase; substrate specificity.

Figure 1.

The Akt2 W80A mutation confers MK2206 resistance and has negligible impact on Akt2 activation at the plasma membrane in 3T3-L1 adipocytes. A, the Akt W80A mutation confers resistance to the Akt inhibitor MK2206. Treatment of cells ectopically expressing W80A Akt with MK2206 prevents activation of endogenous Akt, allowing specific activation of the W80A Akt mutant upon insulin stimulation. IR, insulin receptor; P, phosphorylation site. B, 3T3-L1 adipocytes were electroporated with TagRFP-T-WT or W80A Akt2, and plasma membrane recruitment was assessed using live-cell TIRF microscopy. Adipocytes were exposed to 1 nm insulin. Representative images for 3–7 independent experiments are presented. C, quantification of B (WT, 41 cells from three independent experiments; W80A, 60 cells from seven independent experiments; mean ± S.E.). D, 3T3-L1 adipocytes were electroporated with TagRFP-T WT or W80A Akt2, and plasma membrane recruitment was assessed using live-cell TIRF microscopy. Adipocytes were exposed to 10 μm MK2206 for 5 min, followed by 1 nm insulin. Representative images for two independent experiments are presented. E, quantification of D (WT, 15 cells from two independent experiments; W80A, 59 cells from two independent experiments; mean ± S.E.).

Figure 2.

W80A Akt2 is resistant to MK2206 and can be used to study Akt2 mutants in 3T3-L1 adipocytes independent of endogenous Akt. A, FLAG-tagged Akt2 constructs were generated: WT, W80A, W80A-T309A, and W80A-S474A Akt2. Each consisted of mutations in the PH domain, kinase domain (KD), and HM. B, lysates from 3T3-L1 adipocytes stably expressing empty vector (EV), WT, W80A, W80A-T309A, or W80A-S474A Akt2 were immunoblotted using antibodies as specified, with 14-3-3 as a loading control. A representative blot for three independent experiments is shown. C, quantification of FLAG expression in B (n = 3, mean ± S.E.). AU, arbitrary unit. D, quantification of Akt2 expression in B relative to the empty vector cell line, which represents endogenous Akt2 (n = 3, mean ± S.E.). E, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were treated with 10 μm MK2206 for 5 min followed by 1 nm insulin for 10 min. Lysates were immunoblotted with antibodies as specified, with 14-3-3 as a loading control. A representative blot for three independent experiments is presented. Band 1 and Band 2 indicate shifts in Akt2 gel migration. p, phosphorylated. F, quantification of E (n = 3, mean ± S.E.).

Figure 3.

Akt2 Ser⁴⁷⁴ phosphorylation is required for maximal insulin-stimulated phosphorylation of PRAS40, AS160, FOXO1/3a, and TSC2 in 3T3-L1 adipocytes. 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were treated with 10 μm MK2206 for 5 min, followed by 1 nm insulin for the times specified. A, lysates were immunoblotted with antibodies as specified, with 14-3-3 as a loading control. A representative blot for three to four independent experiments is presented. Band 1 and Band 2 indicate shifts in Akt2 gel migration. p, phosphorylated. B, quantification of A (n = 3–4, mean ± S.E., two-way analysis of variance; *, p < 0.05; ns, not significant). AU, arbitrary unit; p, phosphorylated.

Figure 4.

Akt2 Ser⁴⁷⁴ phosphorylation is required for maximal insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake in 3T3-L1 adipocytes. A, Akt facilitates insulin-stimulated GLUT4 translocation, glucose uptake, mTORC1 activation, protein synthesis, and FOXO nuclear exclusion. B, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were treated with 10 μm MK2206 for 5 min, followed by 1 nm insulin for 10 min. Lysates were immunoblotted with antibodies as specified, with 14-3-3 as a loading control. A representative blot for three independent experiments is presented. Band 1 and Band 2 indicate shifts in Akt2 gel migration. p, phosphorylated. C, quantification of B (n = 3, mean ± S.E., two-tailed paired t test; *, p < 0.05). AU, arbitrary unit. D, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were treated with 10 μm MK2206 for 5 min followed by 1 nm insulin for 1 h and assessed for [³H]leucine incorporation for protein synthesis (n = 4, mean ± S.E., two-tailed paired t test; *, p < 0.05). E, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were electroporated with FOXO1-mNeonGreen. Cells were treated with 10 μm MK2206 for 5 min, followed by 1 nm insulin and nuclear exclusion assessed using live-cell spinning-disk confocal microscopy. Representative images for three independent experiments are presented. F, quantification of E (WT, 45 cells from three independent experiments; W80A, 63 cells from three independent experiments; W80A-S474A, 63 cells from three independent experiments; mean ± S.E.). G, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were electroporated with pHluorin-GLUT4-mRuby3. Cells were treated with 10 μm MK2206 for 5 min followed by 1 nm insulin and GLUT4 translocation to the plasma membrane and assessed using live-cell TIRF microscopy. Representative images for three independent experiments are presented. H, quantification of G (WT, 78 cells from three independent experiments; W80A, 138 cells from three independent experiments; W80A-S474A, 135 cells from three independent experiments, mean ± S.E.). I, 3T3-L1 adipocytes stably expressing FLAG-Akt2 mutants were treated with 10 μm MK2206 for 5 min followed by 1 nm insulin for 20 min and assessed for [³H]2-deoxyglucose uptake (n = 4, mean ± S.E., two-tailed paired t test; *, p < 0.05).