Isoprenylation of a protein kinase. Requirement of farnesylation/alpha-carboxyl methylation for full enzymatic activity of rhodopsin kinase.
The primary structure of bovine rhodopsin kinase (RK), which phosphorylates light-activated rhodopsin (Rho*), terminates with the amino acid sequence Cys558-Val-Leu-Ser561, a motif that has been shown to direct the isoprenylation and alpha-carboxyl methylation of many proteins (e.g. p21Ha-ras). Transient expression of RK in COS-7 cells revealed the presence of two immunoreactive protein species. Consistent with RK being modified by isoprenylation, interconversion of these two species was dependent upon isoprenoid biosynthesis in the cells. Moreover, a serine substitution for Cys558 resulted in a single RK species whose migration on sodium dodecyl sulfate-polyacrylamide gels was identical to that of RK from cells treated with mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and, thus, of isoprenoid biosynthesis. This finding indicates that isoprenylation of RK requires Cys558. The electrophoretic mobility of isoprenylated RK synthesized in COS-7 cells was identical to that of RK from bovine rod outer segments, suggesting that RK is isoprenylated in vivo. RK was determined to be modified by a farnesyl moiety and alpha-carboxyl-methylated. A time course of Rho* phosphorylation revealed that non-processed RK is approximately 4-fold less active than wild-type RK. This is the first demonstration of isoprenylation/alpha-carboxyl methylation of a protein kinase, and suggests that these modifications markedly influence enzymatic activity in vivo.
Inglese
J
J
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Glickman
J F
JF
Lorenz
W
W
Caron
M G
MG
Lefkowitz
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008745:Methylation; D008798:Mevalonic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D014162:Transfection
D005136:Eye Proteins; D011993:Recombinant Fusion Proteins; D008715:Methionine; D005982:Glutathione Transferase; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D008798:Mevalonic Acid