Moyers J S, Linder M E, Shannon J D, Parsons S J
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochem J. 1995 Jan 15;305 ( Pt 2)(Pt 2):411-7. doi: 10.1042/bj3050411.
Overexpression of pp60c-src in mouse fibroblasts potentiates both agonist-induced signalling through beta-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin [Bushman, Wilson, Luttrell, Moyers and Parsons (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7462-7466; Moyers, Bouton and Parsons (1993) Mol. Cell. Biol. 13, 2391-2400]. In reconstitution experiments in vitro, phosphorylation of Gs alpha by immune-complexed pp60c-src resulted in enhanced rates of receptor-mediated guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding and GTP hydrolysis [Hausdorff, Pitcher, Luttrell, Linder, Kurose, Parsons, Caron and Lefkowitz (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5720-5724]. These results suggest that one mechanism by which pp60c-src affects signalling through the beta-adrenergic receptor is by phosphorylation and functional alteration of the G protein. To elucidate how phosphorylation of Gs alpha might affect its function, we subjected phosphorylated, recombinant Gs alpha to tryptic phosphopeptide analysis. Phosphotryptic peptides were purified by h.p.l.c. and analysed by Edman degradation to determine the cycle numbers at which radiolabelled phosphotyrosine was released. Candidate peptides that contained Tyr residues at the corresponding positions were synthesized, phosphorylated in vitro by pp60c-src, and their migrations in two-dimensional electrophoresis/t.l.c. were compared with those of tryptic phosphopeptides from intact Gs alpha. We report here that Gs alpha is phosphorylated on two residues by pp60c-src, namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of beta gamma binding in the N-terminus, within a region postulated to modulate GDP dissociation and activation by GTP [Johnson, Dhanasekaran, Gupta, Lowndes, Vaillancourt and Ruoho (1991) J. Cell Biochem. 47, 136-146], while Tyr-377 is located in the extreme C-terminus, within a region of Gs alpha important for receptor interaction [Sullivan, Miller, Masters, Beiderman, Heideman and Bourne (1987) Nature (London) 334, 712-715]. The location of these residues suggests that phosphorylation may affect the function of both of these regulatory domains.
pp60c-src在小鼠成纤维细胞中的过表达增强了激动剂通过β-肾上腺素能受体诱导的信号传导以及对霍乱毒素的环磷酸腺苷积累[布什曼、威尔逊、卢特雷尔、莫耶斯和帕森斯（1990年）《美国国家科学院院刊》87, 7462 - 7466；莫耶斯、布顿和帕森斯（1993年）《分子与细胞生物学》13, 2391 - 2400]。在体外重组实验中，免疫复合物pp60c-src对Gsα的磷酸化导致受体介导的鸟苷5'-[γ-硫代]三磷酸（GTP[S]）结合和GTP水解速率增强[豪斯多夫、皮彻、卢特雷尔、林德、黑濑、帕森斯、卡隆和莱夫科维茨（1992年）《美国国家科学院院刊》89, 5720 - 5724]。这些结果表明，pp60c-src影响通过β-肾上腺素能受体信号传导的一种机制是通过G蛋白的磷酸化和功能改变。为了阐明Gsα的磷酸化如何影响其功能，我们对磷酸化的重组Gsα进行了胰蛋白酶磷酸肽分析。磷酸化胰蛋白酶肽通过高效液相色谱法纯化，并通过埃德曼降解进行分析，以确定释放放射性标记磷酸酪氨酸的循环次数。合成了在相应位置含有酪氨酸残基的候选肽，在体外由pp60c-src进行磷酸化，并将它们在二维电泳/薄层层析中的迁移情况与完整Gsα的胰蛋白酶磷酸肽进行比较。我们在此报告，Gsα被pp60c-src磷酸化在两个残基上，即酪氨酸-37和酪氨酸-377。酪氨酸-37位于N端βγ结合位点附近，在一个假定调节GDP解离和GTP激活的区域内[约翰逊、达纳塞卡兰、古普塔、朗兹、瓦扬古和鲁霍（1991年）《细胞生物化学杂志》47, 136 - 146]，而酪氨酸-377位于极端C端，在Gsα对受体相互作用重要的区域内[沙利文、米勒、马斯特斯、贝德曼、海德曼和伯恩（1987年）《自然》（伦敦）334, 712 - 715]。这些残基的位置表明磷酸化可能影响这两个调节域的功能。
