Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.
The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.
Chen
Y
Y
Centre de Biochimie, CNRS UMR134, Parc Valrose, Nice, France.
Grall
D
D
Salcini
A E
AE
Pelicci
P G
PG
Pouysségur
J
J
Van Obberghen-Schilling
E
E
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D037342:Pertussis Toxin; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013917:Thrombin / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D018179:Receptors, Thrombin; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D010566:Virulence Factors, Bordetella; D005346:Fibroblast Growth Factors; D037342:Pertussis Toxin; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; D013917:Thrombin; D019204:GTP-Binding Proteins; D018631:ras Proteins