COOH-terminal methylation of lamin B and inhibition of methylation by farnesylated peptides corresponding to lamin B and other CAAX motif proteins.
Previous reports from this laboratory have demonstrated that lamin B is reversibly methylesterified in a cell cycle-dependent manner. The site of this methylation, however, was not identified. In this report, we describe a single major methylated product obtained following reversed-phase high-performance liquid chromatographic analysis of peptides generated by proteolytic digestion of lamin B from rat liver nuclear envelopes. This peptide was retained on a lamin B COOH-terminal-specific antibody-affinity column, and COOH-terminal localization was confirmed by amino acid sequencing. Two other COOH-terminal peptides were found but were not methylated and differed in sequence by at least a single residue from the methylated peptide, indicating the existence of two lamin B gene products. Tetrapeptides, representing the putative mature COOH termini of lamin B, K-ras-2A, and unprocessed lamin A, were synthesized with or without farnesyl modification of the COOH-terminal cysteines. All three farnesylated peptides served as substrates for the partially purified lamin B methyltransferase with apparent Km values of 4.5, 0.69, and 21 microM, respectively. Nonfarnesylated peptides were not substrates for the enzyme. The three farnesylated peptides were also effective to varying degrees at inhibiting the methylation of lamin B and other cellular proteins in cell lysates.
Sobotka-Briner
C
C
Cancer Discovery Research, Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328.
Chelsky
D
D
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D005204:Farnesol / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D008099:Liver / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010449:Peptide Mapping; D051381:Rats
D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D009842:Oligopeptides; D005204:Farnesol