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

Kyounghwa Lee,Gil Hong Park,Tae Jin Kim,Kyung-Mi Lee,Kyo Won Lee,Meyoung Kon Kim,Jong Seol Han,Yoon Sik Hong

doi:10.1038/emm.2004.72

Abstract

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.

Highlights

Methylation has been shown to play important roles in several biological phenomena occurring in prokaryotes as well as eukaryotes
Protein methylation is found in the residues, arginine, aspartate, glutamate, lysine and C-terminal cysteine, which has been shown to occur in a variety of cellular processes including protein trafficking, protein-protein interaction, and various signal transduction pathways leading to cellular proliferation (Paik and Kim, 1980; McBride and Silver, 2001)
Earlier studies demonstrated that the activity of protein arginine methyltransferase (PRMT) was directly correlated with the degree of proliferation and it was found to be elevated in highly proliferating tissues such as regenerating liver (Kwon et al, 2004), developing fetal brain, rapidly growing hepatomas and continuously dividing HeLa cells (Paik et al, 1975; Paik and Kim, 1980)

Summary

Introduction

Methylation has been shown to play important roles in several biological phenomena occurring in prokaryotes as well as eukaryotes. Protein methylation is found in the residues, arginine, aspartate, glutamate, lysine and C-terminal cysteine, which has been shown to occur in a variety of cellular processes including protein trafficking, protein-protein interaction, and various signal transduction pathways leading to cellular proliferation (Paik and Kim, 1980; McBride and Silver, 2001). We showed recently that methylation of this cytosolic 20-kDa protein was increased in a more physiologic setting, such as proliferating hepatocytes, due to elevated methyltransferase activity, and that histones were likely to play some regulatory roles in this process (Kwon et al, 2004). In contrast to the cytosolic fraction, which showed persistent methylation of a 20-kD band up to 7 days following hepatectomy, methylation of nuclear substrates do not show any persistent changes in methylation Rather their level of methylation was more or less consistent before or after hepatectomy. Our data describing the characteristics and specificity of the methyltransferases regulating methylation of this 16-kD protein proposes the importance of methylation events in the process of liver regeneration

Materials and Methods

Partial hepatectomy

Preparation of nuclear extracts

Findings

SAH None MBP Cyt ADH Car Glo Lac His