Identification of a dominant-negative mutation in the yeast CDC25 guanine nucleotide exchange factor for Ras.
Identification of a dominant-negative mutation in the yeast CDC25 guanine nucleotide exchange factor for Ras.
复制标题
鉴定 Ras 酵母 CDC25 鸟嘌呤核苷酸交换因子中的显性失活突变。
作者：
Park,W;Mosteller,RD;Broek,D
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 [3 H]-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.
