Characterization and properties of dominant-negative mutants of the Ras-specific guanine nucleotide exchange factor CDC25(Mm)

Res proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of G TPase activating proteins and guanine nucleotide exchange factors (GEF). Th e properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25(Mm), are described. In vitro, the mutant GEF (W1056E) and GEF(T1184E) proteins are catalytically inactive, are able to e fficiently displace wild-type GEF from p21(ras), and strongly reduce affini ty of the nucleotide-free ras GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. Th e stable ectopic expression of the GEF(W1056E) mutant in smooth muscle cell s effectively reduced growth rate and DNA synthesis with no detectable morp hological changes.