The Crk signaling pathway contributes to the bombesin-induced activation of the small GTPase Rap1 in Swiss 3T3 cells.
Rap1 is a small GTPase implicated in cell proliferation and differentiation. The mechanisms how endogenous Rap1 is activated by many mitogenic stimuli including the neuropeptide bombesin remained unclear. Here we analyse which signaling pathways are necessary for Rap1 activation. Bombesin-mediated Rap1 activation in Swiss 3T3 and primary mouse embryo fibroblasts requires signaling components similar to those being essential for complex formation between p130Cas and Crk adapter proteins. The Crk/CRKL-binding region of the Rap1-specific exchange factor C3G (CBR) inhibits the bombesin-stimulated Rap1 activity in transfected Swiss 3T3 cells. Further characterization in COS cells showed that the CBR or a c-Crk I SH3 mutant specifically reduces both the basal as well as the stimulated Rap1 activity in a dose-dependent manner, whereas Ras is not affected. The CBR is complexed with endogenous c-Crk II and CRKL and blocks the protein association with catalytically active C3G. Such suppressors of Crk signaling do not affect Erk-phosphorylation induced by bombesin. Embryonic fibroblasts from b-raf knockout mice showed a bombesin-inducible Erk-phosphorylation, providing evidence that B-Raf does not link Rap1 to Erk-activation in bombesin-stimulated fibroblasts. We conclude that cellular Crk/CRKL complexes, recruited to upstream signaling components, contribute to basal and bombesin-induced Rap1 activity, which is independent from the Ras-Raf-Erk pathway under these circumstances.
Posern
G
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Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Julius-Maximilians University, D-97078 Würzburg, Germany.
Rapp
U R
UR
Feller
S M
SM
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001839:Bombesin / Q000494:pharmacology*; D019556:COS Cells; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000235:genetics / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050737:Proto-Oncogene Proteins c-crk; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D020735:Guanine Nucleotide-Releasing Factor 2; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D020827:rap1 GTP-Binding Proteins; D001839:Bombesin