Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors

We have analyzed in Chinese hamster ovary cells the upstream mediators by w hich the G protein-coupled receptor, gastrin/CCKB, activates the extracellu lar-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc complete ly blocked gastrin-stimulated Shc.Grba complex formation but partially inhi bited ERK-1 activation by this peptide. Expression of Csk, which inactivate s Src-family kinases, totally inhibited gastrin-induced Src-like activity d etected in anti-Src and anti-She precipitates but diminished by 50% Shc pho sphorylation and ERK-1 activation. We observed a rapid tyrosine phosphoryla tion of insulin receptor substrate-1 (IRS-1) and an increase in Src-like ki nase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cell s, 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 format ion and IRS-1 phosphorylation in Csk-transfected cells. In addition, the in crease in PI 3-kinase activity measured in anti p85 or anti-IRS-1 precipita tes following gastrin stimulation was abolished by Csk. Our results demonst rate the existence of two mechanisms in gastrin-mediated ERKs activation, O ne requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosph orylation 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.