Calmodulin-independent coordination of Ras and extracellular signal-regulated kinase activation by Ras-GRF2

Ras-GRF2 (GRFZ) is a widely expressed, calcium-activated regulator of the small-type GTPases Pas and Pac. It is a multidomain protein composed of several recognizable sequence motifs in the following order (NH2 to COOH): pleckstrin homology (PH), coiled-coil, ilimaquinone (IQ), DbI homology (DH), PH, REM (Ras exchanger motif), PEST/destruction box, Cdc25. The DH and Cdc25 domains possess guanine nucleotide exchange factor (GEF) activity and interact with Pac and Pas, respectively. The REM-Cdc25 region was found to be sufficient for maximal activation of Pas in vitro and in vivo caused Pas and extracellular signal-regulated kinase (ERK) activation independent of calcium signals, suggesting that, at least when expressed ectopically, it contains all of the determinants required to access and activate Ras signaling. Additional mutational analysis of GRFZ indicated that the carboxyl PH domain imparts a modest inhibitory effect on Pas GEF activity and probably normally participates in intermolecular interactions. A variant of GRFZ missing the Cdc25 domain did not activate Pas and functions as an inhibitor of wild-type GRF2, presumably by competing for interactions with molecules other than calmodulin, Pas, and ligands of the PH domain. The binding of calmodulin was found to require several amino-terminal domains of GRF2 in addition to the IQ sequence, and no correlation between calmodulin binding by GRFZ and its ability to directly activate Pas and indirectly stimulate the mitogen-activated protein (MAP) kinase ERK in response to calcium was found. The precise role of the GRF2-calmodulin association, therefore, remains to be determined. A GRF2 mutant missing the IQ sequence was competent for Pas activation but failed to couple this to stimulation of the ERK pathway. This demonstrates that Ras-GTP formation is not sufficient for MAP kinase signaling. We conclude that in addition to directly activating Pas, GRFZ, and likely other GEFs, promote the assembly of a protein network able to couple the GTPase with particular effecters.