Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales 2006, Australia
J Biol Chem. 2019 Nov 8;294(45):16729-16739. doi: 10.1074/jbc.RA119.010036. Epub 2019 Sep 22.
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; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser phosphorylation in 3T3-L1 adipocytes by preventing Ser 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 Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser phosphorylation. We propose a model where Ser phosphorylation is required for maximal Akt2 kinase activity in adipocytes.
丝氨酸/苏氨酸蛋白激酶 Akt 调节着细胞存活、生长和代谢等重要的生物学过程。在生长因子刺激下，Akt 在丝氨酸处发生磷酸化；然而，这种磷酸化如何促进 Akt 的激活仍然存在争议。先前的研究通过消除其上游激酶 mTORC2 来促使丝氨酸磷酸化缺失，这些研究表明丝氨酸磷酸化是 Akt 底物特异性的驱动因素。在这里，我们通过在不干扰 mTORC2 活性的情况下直接阻止丝氨酸磷酸化，在 3T3-L1 脂肪细胞中直接研究了 Akt2 丝氨酸磷酸化的作用。这是通过利用化学遗传学方法实现的，其中通过引入 W80A 突变，将异位表达的 S474A Akt2 工程化为对 Akt 抑制剂 MK2206 具有抗性的蛋白，从而使其能够独立于内源性 Akt 被激活。我们发现，在不存在丝氨酸磷酸化的情况下，胰岛素刺激的四种 Akt 底物（TSC2、PRAS40、FOXO1/3a 和 AS160）的磷酸化减少了约 50%。相应地，当丝氨酸磷酸化缺失时，胰岛素刺激的 mTORC1 激活、蛋白质合成、FOXO 核输出、GLUT4 易位和葡萄糖摄取均减弱。我们提出了一个模型，其中丝氨酸磷酸化是脂肪细胞中 Akt2 激酶活性达到最大所必需的。
