Characterization of Ras-GRF2, a bifunctional guanine nucleotide exchange factor for the Ras and Rac GTPases

grantor: University of TorontoActivation of Ras is catalyzed by guanine nucleotide exchange factors, including Ras-GRF2, which was cloned in our laboratory. Ras-GRF2 is a widely expressed protein comprised of a number of modular domains. They include two pleckstrin homology (PH) domains, a coiled-coil motif, an ilimaquinone (IQ) motif, a Dbl-homology (DH) domain and a Cdc25 domain. Here I report the characterization of the IQ motif, the DH domain and the Cdc25 domain, and demonstrate that Ras-GRF2 functions as a calcium-responsive dual-specificity exchange factor for the Ras and Rac GTPases. A recombinant glutathione S-transferase fusion protein containing the Cdc25 domain of Ras-GRF2 stimulated net nucleotide exchange on Ras, ' in vitro'. Consistent with this result, Ras-GRF2 was also able to activate the extracellular signal-regulated kinase (ERK), which functions downstream of Ras. ERK activation was found to be both Ras-dependent and calcium-responsive. The coimmunoprecipitation of Ras-GRF2 and the calcium sensor calmodulin was shown to be dependent on the presence of an intact IQ motif. The calcium-responsive nature of Ras-GRF2 activity may be related to its subcellular localization. In unstimulated cells, transiently expressed Ras-GRF2 proteins appeared cytosolic, whereas in cells stimulated with a calcium ionophore, a relocalization of Ras-GRF2 to the cell periphery was observed. Although the overexpression of Ras-GRF2 in NIH 3T3 and Rat2 cells was not transforming, the stable expression of Ras-GRF2 in 293 epithelial cells altered the growth properties of the cells. GST pulldown experiments revealed that Ras-GRF2 associated with the nucleotide-free forms of Ras and Rac, weakly with Rho, and not at all with Cdc42. The interaction between Ras and Ras-GRF2 required the Cdc25 domain, whereas its association with Rac was mediated by the DH domain. Ras GRF2 was shown to catalyze nucleotide exchange on Rac. Ras-GRF2 also activated the stress activated protein kinase (SAPK) in a calcium-dependent manner. SAPK activation required the IQ and DH domains, and to a lesser extent, the Cdc25 domain. The activation of both ERK and SAPK by Ras-GRF2 could be inhibited by the dominant-negative mutants of Ras or Rac. Full activation of SAPK by Ras-GRF2 appears to involve the activation of Ras by the Cdc25 domain, as expression of the Cdc25 domain led to SAPK activation while a point mutant of Ras-GRF2 that can no longer bind to Ras activates SAPK less effectively than wild-type. The PH domains of Ras-GRF2 do not appear to be involved in SAPK activation. Instead, expression of the DH domain or the amino terminal PH domain-deleted mutant resulted in higher levels of SAPK phosphorylation than that observed with wild-type Ras-GRF2.Ph.D