Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP.
The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25(Mn) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family GTPase Cdc42 through still unknown mechanisms. Here, we report that retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomycin-stimulated activities were unaffected by a constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF performance. Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inhibitory effects on Ras-GRF function. These results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded. Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.
Arozarena
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I
Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain 28029.
Matallanas
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D
Crespo
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P
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020666:ras-GRF1 / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D020730:Guanine Nucleotide Dissociation Inhibitors; D008246:Lysophospholipids; D011994:Recombinant Proteins; D020666:ras-GRF1; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D015759:Ionomycin; D006160:Guanosine Triphosphate; D020928:Mitogen-Activated Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins