Maintenance of Cdc42 GDP-bound State by Rho-GDI Inhibits MAP Kinase Activation by the Exchange Factor Ras-GRF

Abstract

The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25Mn 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 GDPbound 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.Small GTP-binding proteins act as key molecular switches in signal transduction routes through which stimuli received through cell surface receptors are conveyed to the nucleus. The hallmark of small GTP-binding protein function, the transition from an inactive GDP-bound state to an activated GTP-bound state, is tightly regulated by three types of regulatory proteins: GTPase-activating proteins (GAPs), 1 potentiators of the intrinsic capacity of small G proteins to hydrolyze GTP; guanine nucleotide dissociation inhibitors (GDIs) that regulate the nucleotide interchange process by preventing GDP release; and guanine nucleotide exchange factors (GEFs) that initiate the nucleotide replacement cycle by catalyzing the exchange of GDP for GTP (1).Ras-GRF/Cdc25 mn is a GEF for the Ras family of small GTP-binding proteins cloned by virtue of its homology with the CDC25 gene product that stimulates nucleotide exchange on Saccharomyces cerevisiae Ras (2-5). The primary structure of Ras-GRF reveals the presence of a number of regulatory motifs presumably involved in diverse signaling control mechanisms and protein-protein interactions. These include a Dbl homology (DH) domain (6), generally present in GEFs for the Rho family of small G proteins (7). The DH domain is flanked by two Pleckstrin homology (PH) domains, the function of which remains largely unclear, although it has been suggested that it may play a role in membrane targeting (8).With regard to regulation, it is known that Ras-GRF exchange activity over Ras is augmented by G protein-coupled receptors but is largely unaffected by receptors of the tyrosine kinase type (9 -13). Calcium can also regulate Ras-GRF activ...

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Regulation of Protein Transport from the Golgi Complex to the Endoplasmic Reticulum by CDC42 and N-WASP

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