The nuclear 16-kD protein methylation increases in the early period of liver regeneration in a hepatectomized rat

Methylation events play a critical role in various cellular processes including regulation of gene transcription and proliferation. We observed that methyltransferase activity underwent time-dependent changes in the cytosol of the rat hepatocytes upon partial hepatectomy. However, any change in the methylation of nuclear proteins is not clear during hepatocyte proliferation. The nuclear fraction possesses basal level of methyltransferase to catalyze methylation of several proteins ranging from 7 to 70 kD prior to any hepatecmony. The specific p16 (16 kD) band was transiently and heavily methylated post 1 day hepatectomy, and then became non- detectable, but not in the control liver. Methylation of p16 band was completely inhibited by exogenously added histones, particularly 2AS, 1, 2A and 2B subtypes. The methylated p16 protein remains stable in either acid or alkali- induced demethylation conditions, indicating that methylation is not likely to occur on isoaspartyl or C-terminal cysteinyl residues. Exogenous addition of non-hydrolyzable GTP caused a dose- dependent suppression of a p16 methylation suggesting that G-proteins might play a role as an endogenous methylation inhibitor in vivo. Taken together, we have identified the proliferation event associated-methylation of the nuclear p16 protein in the hepatocytes undergoing liver regeneration.

Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain

C-terminal farnesyl cysteine carboxyl methylation has been known to be the last step in the post-translational modification processes of several important signal transduction proteins in eukaryotes including ras related GTP binding proteins and the gamma-subunit of heterotrimeric G proteins. Protein farnesyl cysteine carboxyl methyltransferase (PFCCMT; EC, 2.1.1.100) catalyzing the reaction is well characterized as being stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and suppressed by N-acetyl-S-farnesyl-L-cysteine (AFC). As an initial step to understand the physiological significance of the process, we attempted to purify the enzyme, which was partially purified 130-fold (specific activity, 143 pmol of methyl group transferred/min/mg of protein) with yield of 1.8% after purification by fast protein liquid chromatography (FPLC) on a Superdex 75 column. The enzyme was further purified with non denaturing polyacrylamide gel electrophoresis (ND-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of PFCCMT was determined to be about 30 kDa based on Superdex 75 FPLC as well as photoaffinity labelling with S-adenosyl-L-[methyl-3H] methionine ([methyl-3H]SAM). The partially purified enzyme (Superdex 75 eluate) was found to be characteristically affected by GTP gamma S, being activated about 40-fold in 2 mM, in contrast to ATP which did not show any effect on enzyme activity. Meanwhile, the enzyme was found to be markedly inhibited by AFC, reaching 0 activity in 2 mM. These observations strongly suggested that the partially purified enzyme was PFCCMT.