School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 2011 Jul 15;286(28):24553-60. doi: 10.1074/jbc.M110.202341. Epub 2011 May 18.
Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.
蛋白激酶 B（PKB，Akt）是一种丝氨酸/苏氨酸激酶，参与细胞存活、增殖和代谢的调节，其活性通过在激活环中的 Thr（308）和疏水模体中的 Ser（473）的双磷酸化而被激活。它在血小板功能中发挥辅助作用，尽管对其调节知之甚少。在这项研究中，我们使用最近鉴定的小分子 ATP 竞争性 mTOR 抑制剂 PP242 和 Torin1 研究了哺乳动物雷帕霉素靶蛋白复合物（mTORC）-2 在 Akt 调节中的作用。PP242 和 Torin1 均以 1 至 5nm 的 IC50 阻断凝血酶和胰岛素样生长因子 1 介导的 Akt Ser（473）磷酸化，而 mTORC1 抑制剂雷帕霉素则没有作用。有趣的是，PP242 和 Torin1 对 Akt Thr（308）磷酸化、Akt1 活性和 Akt 底物糖原合酶激酶 3β的磷酸化没有影响，表明 Ser（473）磷酸化不是 Thr（308）磷酸化和最大 Akt1 活性所必需的。相反，在 PP242 和 Torin1 的存在下，Akt2 活性显著降低，同时 PRAS40 磷酸化被抑制。其他信号通路，包括磷脂酶 C/蛋白激酶 C 和 MAPK 通路，不受 PP242 和 Torin1 的影响。综上所述，这些结果表明 mTORC2 是磷酸化人血小板中 Akt Ser（473）的激酶，但这种磷酸化对于 Thr（308）磷酸化和 Akt1 活性是可有可无的。
