Moyers J S, Bouton A H, Parsons S J
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Mol Cell Biol. 1993 Apr;13(4):2391-400. doi: 10.1128/mcb.13.4.2391-2400.1993.
Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.
此前我们证明，与对照细胞相比，过表达禽源c-src的C3H10T1/2小鼠成纤维细胞在受到β-肾上腺素能激动剂刺激时，其环磷酸腺苷（cAMP）水平会升高，且这种增强的反应需要c-src激酶活性（W. A. 布什曼、L. K. 威尔逊、D. K. 卢特雷尔、J. S. 莫耶斯和S. J. 帕森斯，《美国国家科学院院刊》87:7462 - 7466, 1990）。然而，目前尚不清楚β-肾上腺素能受体途径中的哪些成分（如果有的话）与pp60c-src相互作用。最近有研究表明，pp60c-src的免疫复合物在体外能将重组Gα蛋白磷酸化至化学计量水平，从而导致GTP结合和GTP酶活性的改变（W. P. 豪斯多夫、J. A. 皮彻、D. K. 卢特雷尔、M. E. 林德、H. 黑濑、S. J. 帕森斯、M. G. 卡隆和R. J. 莱夫科维茨，《美国国家科学院院刊》89:5720 - 5724, 1992），这增加了Gsα蛋白可能是pp60c-src在体内相互作用靶点的可能性。为了进一步阐明pp60c-src在β-肾上腺素能信号通路中的作用，我们在10T1/2细胞中过表达了pp60c-src，这些pp60c-src在几个被认为对信号传导过程很重要的结构域中存在突变。在本研究中，我们发现蛋白激酶C（PKC）的磷酸化位点（Ser-12和Ser-48）以及pp60c-src的SH2区域对于c-src过表达细胞对β-激动剂刺激的增强反应是必需的。肉豆蔻酰化位点（Gly-2）的突变导致增强反应减弱，而cAMP依赖性蛋白激酶的磷酸化位点（Ser-17）的突变则没有影响。二维磷酸化胰蛋白酶分析表明，未受刺激细胞中Ser-12和Ser-48的磷酸化与过表达的pp60c-src增强β-肾上腺素能信号传导的能力有关。过表达野生型c-src的细胞在用霍乱毒素处理后也表现出cAMP积累增强，而在过表达激酶或SH2结构域有缺陷或PKC磷酸化位点发生改变的pp60c-src的细胞中，这种效应减弱。这些研究首次证明了PKC对pp60c-src磷酸化位点的生理意义。此外，它们表明SH2、Ser-12/48和肉豆蔻酰化区域对于pp60c-src与β-肾上腺素能途径成分的有效相互作用可能很重要。我们的数据也支持Gsα蛋白可能是pp60c-src在体内改变的靶点这一可能性。
