Identification of a dominant-negative mutation in the yeast CDC25 guanine nucleotide exchange factor for Ras

Abstract

In previous studies we changed five conserved amino acid residues in the catalytic domain of the yeast Ras-specific guanine nucleotide exchange factor CDC25^GEF (Park et al., 1994). One of the substitutions (R1489E) resulted in a molecule which could bind Ras but was catalytically inactive. These observations suggested that CDC25^R1489E might be a dominant-negative mutant. Here we report further experiments which confirm the dominant-negative phenotype of CDC25^R1489E. Two lines of evidence indicate that the CDC25^R1489E mutant exhibits Ras-specific binding in vivo. First, expression of CDC25^R1489E in a wild-type yeast strain caused a partial inhibition of growth which was reversed by overexpression of the wild-type yeast RAS2 protein. Second, expression of CDC25^R1489E in a yeast strain containing a temperature-sensitive, dominant-negative RAS2 mutation (RAS2^val19ala22) suppressed the temperature-sensitive phenotype. The latter findings suggest that the CDC25^R1489E protein bound the mutant RAS2 protein thereby releasing the wild-type CDC25 protein for activation of the wild-type RAS1 protein. Further, using a protein-protein binding assay and guanine nucleotide exchange assay (release of [³H]-GDP) in vitro, we demonstrate that the CDC25^R1489E protein can bind wild-type Ras protein but is unable to catalyze GDP-GTP exchange. Thus, the results of genetic and biochemical experiments demonstrate that CDC25^R1489E encodes a dominant-negative GEF which blocks the Ras signaling pathway by binding wild-type Ras in a catalytically inactive complex.