Cholecystokinin stimulates formation of shc-grb2 complex in rat pancreatic acinar cells through a protein kinase C-dependent mechanism.
Cholecystokinin (CCK) has recently been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Ras-Raf-MAPK kinase-MAPK) in pancreatic acini. The mechanism by which the Gq protein-coupled CCK receptor activates Ras, however, is currently unknown. Growth factor receptors are known to activate Ras by means of adaptor proteins that bind to phosphotyrosine domains. We therefore compared the effects of CCK and epidermal growth factor (EGF) on Tyr phosphorylation of the adaptor proteins Shc and its association with Grb2 and the guanine nucleotide exchange factor SOS. Three major isoforms of Shc (p46, p52, p66) were detected in isolated rat pancreatic acini with p52 Shc being the predominant form. CCK and EGF increased tyrosyl phosphorylation of Shc (251 and 337% of control, respectively). CCK-stimulated tyrosyl phosphorylation of Shc as well as Shc-Grb2 complex formation was significant at 2.5 min, maximal at 5 min, and persisted for at least 30 min. Finally, SOS was found to be associated with Grb2 as assessed by probing of anti-Grb2 immunoprecipitates with anti-SOS. Since MAPK in pancreatic acini is activated via protein kinase C (PKC), we studied the effect of phorbol esters on Shc phosphorylation and found 12-O-tetradecanoylphorbol-13-acetate to be as potent as CCK. Furthermore, GF-109203X, a PKC inhibitor, abolished the effect of 12-O-tetradecanoylphorbol-13-acetate and also the effect of CCK but not the effect of EGF on Shc tyrosyl phosphorylation. CCK-induced tyrosyl phosphorylation of Shc was found to be phosphatidylinositol 3-kinase-independent, and CCK did not cause EGF receptor activation. These results suggest that formation of an Shc-Grb2-SOS complex via a PKC-dependent mechanism may provide the link between Gq protein-coupled CCK receptor stimulation and Ras activation in these cells.
Dabrowski
A
A
Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622, USA.
VanderKuur
J A
JA
Carter-Su
C
C
Williams
J A
JA
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002766:Cholecystokinin / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D051379:Mice; D010179:Pancreas / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011949:Receptors, Cholecystokinin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020663:ras Guanine Nucleotide Exchange Factors
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011949:Receptors, Cholecystokinin; D020663:ras Guanine Nucleotide Exchange Factors; D014443:Tyrosine; D004815:Epidermal Growth Factor; D002766:Cholecystokinin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins