Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid.
ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by [3H]geranylgeranyl instead of [3H]farnesyl in an in vitro assay. Gel-permeation chromatography of [3H]mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21(Leu-189) was also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21(Leu-189) with [3H]palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases.
Weis Center for Research, Geisinger Clinic, Danville, PA 17822.
B T
BT
Kinsella
R A
RA
Erdman
W A
WA
Maltese
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine; D019204:GTP-Binding Proteins / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism; D012717:Sesquiterpenes; D014162:Transfection
D009841:Oligonucleotides; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D003545:Cysteine; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid