Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors
Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors
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DOI：
10.1074/jbc.274.29.20657
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发表时间：
1999-07-16
影响因子：
4.8
通讯作者：
Seva, C
中科院分区：
文献类型：
--
作者：
Daulhac, L;Kowalski-Chauvel, A;Seva, C
我们分析了中国仓鼠卵巢细胞的上游介质，G蛋白偶联受体，胃泌素/CCKB，激活细胞外调节激酶（ERK）和p85/p110-磷脂酰肌醇3-激酶（PI 3-激酶）途径。过表达的抑制性突变体的Shc完全阻断胃泌素刺激的Shc。Grba复合物的形成，但部分抑制ERK-1激活的肽。Csk的表达，失活Src家族激酶，完全抑制胃泌素诱导的Src样活性检测抗Src和抗She沉淀，但减少了50%的Shc磷酸化和ERK-1激活。我们观察到胰岛素受体底物-1（IRS-1）的快速酪氨酸磷酸化和抗IRS-1免疫沉淀物中Src样激酶活性的增加，表明IRS-1可能是Src的直接底物。这一假设得到了Csk转染细胞中胃泌素诱导的Src IRS-1复合物形成和IRS-1磷酸化的抑制的支持。此外，增加PI 3-激酶活性测定抗p85或抗IRS-1沉淀后胃泌素刺激被取消的Csk。我们的研究结果表明胃泌素介导的ERK激活存在两种机制，一种需要Src家族激酶磷酸化Shc，另一种不依赖于这两种蛋白。它们还表明Src家族激酶对IRS-1的酪氨酸磷酸化可导致p85/p110-PI 3-激酶的募集和激活以响应胃泌素。
We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grba complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-She precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation, One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.