Publications

• Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate.

  Stephenson R.C., Clarke S.

  Polypeptides synthesized in eucaryotic cells with a C-terminal -Cys-Xaa-Xaa-Xaa (-CXXX) sequence are candidates for post-translational modifications that include the removal of the last 3 amino acids and the lipidation and methyl esterification of the cysteinyl residue. To characterize the methyla ... >> More

  Polypeptides synthesized in eucaryotic cells with a C-terminal -Cys-Xaa-Xaa-Xaa (-CXXX) sequence are candidates for post-translational modifications that include the removal of the last 3 amino acids and the lipidation and methyl esterification of the cysteinyl residue. To characterize the methylation reaction in vitro, the peptide Leu-Ala-Arg-Tyr-Lys-Cys (LARYKC) and its S-isoprenylated and S-alkylated derivatives were synthesized and assayed as methyl-accepting substrates with subcellular fractions of rat tissues including liver microsomal membranes. While little or no peptide-specific methyltransferase activity was detected in the latter preparation using the unmodified hexapeptide, the C10, C15, and C20 isoprenylated derivatives were substrates with Km values of 389 microM for S-geranyl-LARYKC, 2.2 microM for S-farnesyl-LARYKC, and approximately 10.9 microM for S-geranylgeranyl-LARYKC. The methyl-acceptor activities of a variety of n-alkyl S-derivatives of LARYKC (C8, C10, C13, C15) were also tested; all of these compounds were poorer substrates than the S-geranyl derivative. This enzyme activity uses S-adenosyl-L-methionine as the methyl donor (Km = 2.1 microM) and can be inhibited by S-adenosylhomocysteine (Ki = 9.2 microM), a product of the methylation reaction. The S-farnesyl-LARYKC peptide can inhibit the carboxyl methylation of bovine retinal rod outer segment membrane proteins that was previously shown to occur at the alpha-carboxyl group of C-terminal cysteine residues, demonstrating that the same enzyme can methylate both peptides and proteins. These results suggest that the methyl esterification of proteins containing a C-terminal -CXXX sequence requires not only the removal of the 3 terminal amino acids, but the isoprenylation of the sulfhydryl group as well. << Less

  J Biol Chem 265:16248-16254(1990) [PubMed] [EuropePMC]

• Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.

  Clarke S., Vogel J.P., Deschenes R.J., Stock J.

  The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-re ... >> More

  The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the alpha-carboxyl group is also methyl esterified. To determine if ras proteins are themselves modified by a similar type of methylation reaction, we incubated rat embryo fibroblasts transformed with p53 and activated Ha-ras oncogenes with L-[methyl-3H]methionine under conditions in which the isotope was converted to the methyl donor S-adenosyl-L-[methyl-3H]methionine. By using an assay that detects methyl ester linkages, we found that immunoprecipitated ras proteins are in fact esterified and that the stability of these esters is consistent with a carboxyl-terminal localization. This methylation reaction may be important in regulating the interaction of ras proteins with plasma membrane components. The presence of analogous carboxyl-terminal tetrapeptide sequences in other proteins may provide a general recognition sequence for lipidation and methylation modification reactions. << Less

  Proc. Natl. Acad. Sci. U.S.A. 85:4643-4647(1988) [PubMed] [EuropePMC]

• Enzymatic methylation of 23-29-kDa bovine retinal rod outer segment membrane proteins. Evidence for methyl ester formation at carboxyl-terminal cysteinyl residues.

  Ota I.M., Clarke S.

  A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-is ... >> More

  A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77). To determine the site(s) of methylation, 3H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins (Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647). << Less

  J Biol Chem 264:12879-12884(1989) [PubMed] [EuropePMC]