Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes.
Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.
Okada
S
S
Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242, USA.
Matsuda
M
M
Anafi
M
M
Pawson
T
T
Pessin
J E
JE
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D020668:rap GTP-Binding Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D011494:Protein Kinases; D066246:ErbB Receptors; D011972:Receptor, Insulin; D019908:Proto-Oncogene Proteins c-raf; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins