Farnesylation of p21 Ras proteins in Xenopus oocytes.
Unprocessed p21 Ras proteins microinjected into Xenopus oocytes were radiolabeled by coinjected [3H]farnesyl pyrophosphate, a direct farnesyl donor substrate for all known mammalian farnesyltransferases. Mevinolin, an inhibitor of HMG CoA reductase which reduces the levels of mevalonate and thus farnesyl pyrophosphate, blocked oncogenic H-Rasva112 induced germinal vesicle breakdown in oocytes. This mevinolin caused block was completely reversed by co-injected farnesyl pyrophosphate. The putative farnesyltransferase in Xenopus oocytes was identified to be similar to those found in mammalian cells in that it requires an intact CAAX box motif in addition to the conserved cysteine residue at the fourth position from the C-terminus of Ras proteins for its farnesylating activity. Peptide inhibitors of farnesyltransferase such as CVIM and TKCVIM were shown to inhibit farnesylation of microinjected Ras proteins thereby blocking its function namely the induction of oocyte maturation. These results demonstrate that Xenopus oocytes process bacterially produced mammalian Ras proteins in a manner similar to, if not identical with that in mammalian cells, thus validating the continued use of the Xenopus oocyte system for unraveling the functions of Ras proteins. Furthermore, our results indicate that the oocyte system may be a useful in vivo model for studying the farnesylation of human Ras proteins, its regulation, and the effects of farnesyltransferase inhibitors.
Zhao
J
J
Laboratory of Biochemical Physiology, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702-1201.
Kung
H F
HF
Manne
V
V
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D051231:Farnesyltranstransferase; D005260:Female; D007700:Kinetics; D008148:Lovastatin / Q000494:pharmacology; D008322:Mammals; D008798:Mevalonic Acid / Q000378:metabolism; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes; D009866:Oogenesis / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D017368:Protein Prenylation / Q000187:drug effects; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012717:Sesquiterpenes; D014166:Transferases / Q000378:metabolism*; D014316:Tritium; D014982:Xenopus laevis
D010455:Peptides; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D014316:Tritium; C004808:farnesyl pyrophosphate; D008148:Lovastatin; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D016283:Proto-Oncogene Proteins p21(ras); D008798:Mevalonic Acid