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

• Published in: Oncogene Vol. 14; no. 7; pp. 831 - 836
• Main Authors: PARK, W, MOSTELLER, R. D, BROEK, D
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 20.02.1997; Nature Publishing Group
• Subjects: Amino acids; Biological and medical sciences; Cdc25 protein; Cell Cycle Proteins - genetics; Cell Cycle Proteins - physiology; Classical genetics, quantitative genetics, hybrids; Fundamental and applied biological sciences. Psychology; Fungal Proteins - physiology; Genetics of eukaryotes. Biological and molecular evolution; Guanine; Guanine nucleotide exchange factor; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - metabolism; Kinases; Mutants; Mutation; Phenotype; Phenotypes; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - physiology; Proteins; Ras protein; ras Proteins - physiology; ras-GRF1; Ras1 protein; Saccharomyces cerevisiae; Signal Transduction; Thallophyta, bryophyta; Vegetals; Yeast; Fungi; Ligand binding; Yeast; Gene; Molecular interaction; Ascomycetes; Mutation; Guanine nucleotide exchange factor; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1200893

In previous studies we changed five conserved amino acid residues in the catalytic domain of the yeast Ras-specific guanine nucleotide exchange factor CDC25GEF (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 CDC25R1489E might be a dominant-negative mutant. Here we report further experiments which confirm the dominant-negative phenotype of CDC25R1489E. Two lines of evidence indicate that the CDC25R1489E mutant exhibits Ras-specific binding in vivo. First, expression of CDC25R1489E 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 CDC25R1489E in a yeast strain containing a temperature-sensitive, dominant-negative RAS2 mutation (RAS2val19ala22) suppressed the temperature-sensitive phenotype. The latter findings suggest that the CDC25R1489E 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 CDC25R1489E 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 CDC25R1489E encodes a dominant-negative GEF which blocks the Ras signaling pathway by binding wild-type Ras in a catalytically inactive complex.