Ballou Lisa M, Lin Hong-Ying, Fan Gaofeng, Jiang Ya-Ping, Lin Richard Z
Research Service, Department of Veterans Affairs Medical Center, Northport, New York 11768, USA.
J Biol Chem. 2003 Jun 27;278(26):23472-9. doi: 10.1074/jbc.M212232200. Epub 2003 Apr 18.
Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.
一些Gq偶联受体已被证明可拮抗磷脂酰肌醇3激酶（PI3K）及其下游效应分子Akt的生长因子激活。我们使用组成型活性Gαq（Q209L）突变体来探究Gαq激活对通过PI3K/Akt途径的信号传导的影响。Gαq（Q209L）在大鼠-1成纤维细胞中的瞬时表达抑制了血小板衍生生长因子或胰岛素处理诱导的Akt激活。Gαq（Q209L）的表达也减弱了在人胚肾293细胞中组成型活性PI3K共表达所促进的Akt激活。Gαq（Q209L）对两个调节性磷酸化位点被替换为酸性氨基酸的Akt突变体的活性没有影响。在稳定转染的293细胞系中诱导表达Gαq（Q209L）导致p110α（而非p110β）免疫沉淀物中的PI3K活性降低。Gαq的受体激活也选择性抑制了p110α免疫沉淀物中的PI3K活性。活性Gαq在磷脂酶C抑制剂U73122预处理的细胞中仍抑制PI3K/Akt。最后，Gαq（Q209L）与来自表达这些蛋白质的COS-7细胞裂解物中的p110α-p85α PI3K异二聚体共免疫沉淀，并且在体外将免疫沉淀的Gαq（Q209L）与纯化的重组p110α-p85α一起孵育导致PI3K活性降低。这些结果表明激动剂与Gq偶联受体结合通过释放抑制PI3K的活性Gαq亚基来阻断Akt激活。抑制机制似乎独立于磷脂酶C激活，并且可能涉及Gαq与p110α PI3K之间的抑制性相互作用。
