title: Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange.
abstract: We have studied whether hSOS1, a mammalian guanine nucleotide exchange factors responsible for activating Ras in response to growth factor stimulation, requires post-translational processing of Ras proteins to promote guanine nucleotide exchange. Our results showed that full-length hSOS1 catalyzed guanine nucleotide exchange on prenylated K-Ras(4B) but with a much lower efficiency on unprocessed K-Ras(4B). The apparent Km of hSOS1 for prenylated K-Ras(4B) was 225 (+/- 25) nM with a Vmax of 0.7 (+/- 0.1) mmol/min/mmol. The activity of hSOS1 against unprocessed K-Ras(4B) was too low to measure Km and Vmax. Consistent with these observations, full-length hSOS1 formed a complex with nucleotide-depleted prenylated K-Ras(4B) but not with unprocessed K-Ras(4B). A geranylgeranylated mutant of K-Ras(4B) was an equally good substrate for hSOS1 as wild-type farnesylated K-Ras. Similarly hSOS1 promoted guanine nucleotide exchange on prenylated Ha-Ras but showed minimal activity toward unprocessed Ha-Ras. Neither the polybasic domain of K-Ras (4B) or palmitoylation of Ha-Ras were required for hSOS1-promoted guanine nucleotide exchange. We attempted to identify a minimal region of hSOS1 capable of promoting guanine nucleotide exchange on both prenylated and unprocessed K-Ras. However, a truncated form of hSOS1 comprising only the CDC25 homology domain retained preferential catalytic activity against prenylated K-Ras, whereas the cognate domain from CDC25 was more active against unprocessed K-Ras.
authors.affiliation: ONYX Pharmaceuticals, Richmond, California 94806.
authors.forename: E
authors.initials: E
authors.lastname: Porfiri
authors.forename: T
authors.initials: T
authors.lastname: Evans
authors.forename: P
authors.initials: P
authors.lastname: Chardin
authors.forename: J F
authors.initials: JF
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D009036:Moths; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D020663:ras Guanine Nucleotide Exchange Factors
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D010169:Palmitic Acids; D011506:Proteins; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D019308:Palmitic Acid; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras)