Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated.
Rap proteins comprise a subset of the large family of ras-related proteins. They contain the C-terminal tetrapeptide sequence motif Cys-Ali-Ali-Xaa (Ali is an aliphatic amino acid and X is any amino acid), which has been found to be the site of membrane attachment via isoprenylation for ras, nuclear lamins and the gamma subunits of the heterotrimeric G-proteins. To investigate the isoprenylation of rap2a and rap2b, human cDNAs coding for these proteins were expressed in COS cells incubated in the presence of [3H]mevalonolactone. Both proteins incorporated a product of [3H]mevalonolactone, as judged by Western blot analysis. To identify the specific isoprenoid attached to each protein, the cDNAs were transcribed in vitro and the rap2 specific RNA was translated in a rabbit reticulocyte lysate system in the presence of [3H]mevalonolactone. The translation products were treated with methyl iodide and the released isoprenoid groups were analysed by h.p.l.c. Rap2b, which terminates in Cys-Val-Ile-Leu, is geranylgeranylated as predicted while rap2a, which terminates in Cys-Asn-Ile-Gln, incorporated farnesyl. A mutant construct generated by site-directed mutagenesis of rap2a cDNA yielding a protein terminating in leucine instead of glutamine incorporated geranylgeranyl, lending further support to the notion that isoprenoid specificity is governed by the terminal amino acid. In addition, when the CAAX motif cysteine at position 180 of rap2a was replaced by a serine residue no isoprenoid incorporation was observed. Thus rap2a and rap2b, despite showing 90% sequence identity, incorporate different isoprenoid groups. Thus glutamine is a signal for farnesylation, and rap2a is the first non-ras member of the ras superfamily that is farnesylated.
Farrell
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FX
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Yamamoto
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K
Lapetina
E G
EG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D011905:Genes, ras; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008798:Mevalonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D020668:rap GTP-Binding Proteins
D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; C015367:mevalonolactone; D008715:Methionine; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D008798:Mevalonic Acid