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 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.Grb2 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-Shc 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.
Groupe de Recherche de Biologie et Pathologie Digestives, INSERM U. 151, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Batiment L3, 31403 Toulouse, France.
L
L
Daulhac
A
A
Kowalski-Chauvel
L
L
Pradayrol
N
N
Vaysse
C
C
Seva
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006224:Cricetinae; D004261:DNA Replication; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D005755:Gastrins / Q000378:metabolism; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D005755:Gastrins; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011506:Proteins; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases