Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits
Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits
复制标题
DOI：
10.1074/jbc.m007403200
复制
发表时间：
2000-12-08
影响因子：
4.8
通讯作者：
Simonds, WF
中科院分区：
文献类型：
--
作者：
Bommakanti, RK;Vinayak, S;Simonds, WF
虽然受体酪氨酸激酶对c-Akt（也称为蛋白激酶B）的正调节已得到充分证实，但通过G蛋白偶联受体起作用的化合物也可激活Akt及其下游靶标。因此，我们探索了G蛋白亚基在培养的哺乳动物细胞中调节Akt的作用。在分别用β（2）-肾上腺素能或m2毒蕈碱受体瞬时转染的HEK-293和COS-7细胞中，用激动剂诱导的Akt在丝氨酸473处的磷酸化处理，如磷酸丝氨酸特异性免疫印迹所证明的。这种作用被磷脂酰肌醇-3-OH激酶抑制剂LY 294002和野生型G α（IL）阻断，并且不能被组成型活性G α（s）-Q227 L或G α（i）-Q204 L突变体的共转染复制。当在体外对表位标记的酶进行免疫沉淀分析时，G β（2）而不是G β（5）与G γ（2）一起激活激酶。相比之下，组成型激活的G蛋白亚基代表的四个G α亚家族被发现无法激活Akt在任何细胞系。后者的结果与Murga等人的报告（Murga，C.，拉吉湖，韦茨克河，Cuadrado，A.，和Gutkind，J.S.（1998）J，Biol.Chem.273，19080-19085），其描述了在COS细胞中Akt响应于突变激活的G α（q）和G α（i）转染的激活。相反，在我们的实验中，G alpha（q）-Q209 L抑制这些细胞中由β γ或突变激活的H-Ras共转染引起的Akt激活。在HEK-293细胞中，G alpha（q）-Q209 L转染抑制胰岛素样生长因子-1活化表位标记的Akt。在m1毒蕈碱受体转染的HEK-293细胞中，卡巴胆碱以阿托品可逆的方式抑制胰岛素样生长因子-1刺激的内源性Akt的Ser（473）磷酸化。我的结论是，G蛋白可以通过两种不同的和潜在的相反的机制调节Akt：通过G β γ异二聚体以磷脂酰肌醇-3-OH激酶依赖的方式激活，和通过G α（q）介导的抑制。这项工作确定Akt作为不同信号通路之间的一个新的汇合点。
While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in cultured mammalian cells,In HEK-293 and COS-7 cells transiently transfected with beta (2)-adrenergic or m2 muscarinic receptors, respectively, treatment with agonist-induced phosphorylation of Akt at serine 473 as evidenced by phosphoserine-specific immunoblots. This effect was blocked by the phosphatidylinositol-3-OH kinase inhibitor LY294002 and wild-type G alpha (il), and was not duplicated by co-transfection of the constitutively active G alpha (s)-Q227L or G alpha (i)-Q204L mutant, Co-transfection of G beta (1), G beta (2) but not G beta (5) together with G gamma (2) activated the kinase when assayed in vitro following immunoprecipitation of the epitope-tagged enzyme. In contrast, constitutively activated G protein subunits representing the four G alpha subfamilies were found unable to activate Akt in either cell line. The latter results are in disagreement with a report by Murga et al. (Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J, Biol. Chem. 273, 19080-19085) that described activation of Akt in response to mutationally activated G alpha (q) and G alpha (i) transfection in COS cells. To the contrary, in our experiments G alpha (q)-Q209L inhibited Akt activation resulting from beta gamma or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells G alpha (q)-Q209L transfection inhibited insulin-like growth factor-1 activation of epitope-tagged Akt. In m1 muscarinic receptor transfected HEK-293 cells, carbachol inhibited insulin-like growth factor-1 stimulated phosphorylation at Ser(473) of endogenous Akt in an atropine-reversible fashion. me conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by G beta gamma heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by G alpha (q). This work identifies Akt as a novel point of convergence between disparate signaling pathways.