Cloning and analysis of human cDNAs encoding a 140-kDa brain guanine nucleotide-exchange factor, Cdc25GEF, which regulates the function of Ras.
Ras proteins bound to GDP are biologically inactive while those bound to GTP are active. Ras-specific guanine nucleotide-exchange factors (GEFs) have been shown to activate Ras proteins. We used oligodeoxyribonucleotide primers with sequences similar to the cDNAs of rat and mouse cdc25 (encoding a Ras-GEF) to amplify, by the PCR, sequences with the potential to encode a 1275-amino-acid protein homologous to the rodent Cdc25GEF proteins. Northern blot analysis detected a brain-specific 5-kb transcript. We provide evidence for a novel alternately spliced transcript of cdc25 and show that these alternately spliced transcripts are differentially expressed in various regions of the adult nervous system. Antibodies raised against the C terminus of the protein recognize a 140-kDa protein in brain extracts of human, rat, guinea pig and cow; the 140-kDa protein is associated predominantly, if not exclusively, with a crude membrane fraction of brain. The C terminus of human Cdc25GEF can complement the loss of CDC25 function in Saccharomyces cerevisiae. A glutathione S-transferase fusion protein containing the C terminus of the cdc25 product can stimulate guanine nucleotide exchange on H-Ras in vitro. Further, the Cdc25-fusion protein binds tightly to the nucleotide-free form of H-Ras in vitro, and this binding is reversed by the addition of GTP.
Wei
W
W
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033.
Das
B
B
Park
W
W
Broek
D
D
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis* / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic; D018631:ras Proteins / Q000378:metabolism*; D020666:ras-GRF1
D018076:DNA, Complementary; D011506:Proteins; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins