Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor.
The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure-function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.
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Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain, Montreal, QC, H2X 2P2, Canada.
Fixman
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D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins / Q000494:pharmacology; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin; D018909:src Homology Domains; D019061:src-Family Kinases / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; C098587:GAB1 protein, human; C490897:Gab1 protein, mouse; D009025:Morpholines; D010750:Phosphoproteins; D011506:Proteins; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; C073882:phosphoprotein phosphatase inhibitor 1; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin