ABSTRACT
The best characterized yeast guanine nucleotide releasing factor is CDC25, which acts on RAS and thereby stimulates cAMP production in Saccharomyces cerevisiae. In order to determine if CDC25 could be a specific GDP-GTP releasing factor for the mammalian proteins Ha-ras, Ki-ras, and N-ras, its functions were studied both in vitro and in NIH3T3 cells. The 561 amino acid composing the C-terminal domain of CDC25 (CDC25 C-domain) released guanine nucleotides (both GDP and GTP) from Ha-, Ki-, and N-ras but not from Rap1A, Rab5, and Rab11. CDC25 acted on oncogenically activated Ha-ras even if the last 23 amino acids (167-189) of the Ras proteins were not present. CDC25 transformed NIH3T3 cells; its transforming capacity was enhanced by overexpression of wild-type Ha-ras. CDC25 C-domain probably exerts its effects through the activation of cellular Ras proteins. These data suggest that the CDC25 C-domain can function as an upstream activator of Ras proteins in a heterologous system and therefore could be a useful tool to study the regulation of Ras activation by growth factor receptors.
Subject(s)
Cell Cycle Proteins , Cell Transformation, Neoplastic , Fungal Proteins/metabolism , GTP-Binding Proteins/metabolism , Guanosine Diphosphate/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Saccharomyces cerevisiae/metabolism , ras-GRF1 , 3T3 Cells , Animals , Enhancer Elements, Genetic , Escherichia coli/genetics , Fungal Proteins/genetics , GTP-Binding Proteins/genetics , Guanosine Triphosphate/metabolism , Kinetics , Mice , Mice, Nude , Neoplasm Transplantation , Plasmids , Polyomavirus/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Transfection/methodsABSTRACT
[3H]52770 RP, a PAF antagonist, was found to bind with high affinity and in a reversible manner on specific and saturable binding sites in guinea pig cortical membranes. Scatchard analysis revealed the presence of one class of binding sites with an equilibrium dissociation constant of 0.38 nM and a Bmax of 1190 fmol/mg protein. However, these binding sites did not correspond to the PAF receptors described with this ligand in platelet plasma membranes; indeed, PAF and its analogs were unable to displace [3H]52770 RP binding in guinea pig brain. Therefore, one may conclude that [3H]52770 RP in guinea pig brain labels acceptor or recognition sites, rather than true receptor sites.
