Polyisoprenylation of Ras in vitro by a farnesyl-protein transferase.
Farnesylation of Ras occurs in vivo on a Cys residue in the C-terminal sequence -Cys-Val-Leu-Ser (termed a CAAX box). This modification is required for Ras membrane localization and cell transforming activity. Using [3H]farnesyl-PPi as precursor and Escherichia coli-expressed Ras, forms of Ras having the CAAX sequence were radiolabeled upon incubation with the cytosolic fraction of bovine brain. Forms of Ras having a deletion of the CAAX sequence or a Cys to Ser substitution in this sequence were not substrates. Radioactivity incorporated into Ras by bovine brain cytosol was released by treatment with iodomethane but not with methanolic KOH indicating a thioether linkage. High pressure liquid chromatography analysis of the cleavage products on a C-18 column showed a major peak of radioactivity that co-eluted with a farnesol standard. The enzyme responsible for Ras farnesylation in bovine brain was approximately 190 kDa as estimated by gel filtration and required a divalent cation for activity. Nonradioactive farnesyl-PPi, geranylgeranyl-PPi, and Ras peptides having the C-terminal sequence -Cys-Val-Leu-Ser competed in the assay with IC50 values of 0.7, 1.4, and 1-3 microM, respectively. Farnesol and Ras peptides having the sequence -Ser-Val-Leu-Ser were not inhibitory. These results identify a farnesyl-protein transferase activity that may be responsible for the polyisoprenylation of Ras in intact cells.
Schaber
M D
MD
Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
O'Hara
M B
MB
Garsky
V M
VM
Mosser
S C
SC
Bergstrom
J D
JD
Moores
S L
SL
Marshall
M S
MS
Friedman
P A
PA
Dixon
R A
RA
Gibbs
J B
JB
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes; D013379:Substrate Specificity; D014166:Transferases
D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D015689:Oncogene Protein p21(ras); D008798:Mevalonic Acid