Characterization and Properties of Dominant-negative Mutants of the Ras-specific Guanine Nucleotide Exchange Factor CDC25Mm

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The 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 efficiently displace wild-type GEF from p21 ras , and strongly reduce affinity of the nucleotide-free rasGEF complex for the incoming nucleotide, thus resulting in the formation of a stable rasGEF 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. The stable ectopic expression of the GEF W1056E mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.