Identification of a dominant-negative mutation in the yeast CDC25 guanine nucleotide exchange factor for Ras.
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.
Park
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Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California, Los Angeles 90033, USA.
Mosteller
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Broek
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D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D005656:Fungal Proteins / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D018631:ras Proteins / Q000502:physiology*; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins