Phosphorylation of methylated-DNA-protein-cysteine S-methyltransferase at serine-204 significantly increases its resistance to proteolytic digestion

Biochem J. 2000 Dec 15;352 Pt 3(Pt 3):801-8.

Abstract

In a previous paper [Lim, Park, Jee, Lee and Paik (1999) J. Cancer Res. Clin. Oncol. 125, 493-499], we showed two major forms of active DNA-6-O-methylguanine:protein-L-cysteine S-methyltransferase (MGMT; EC 2.1.1.63) in the liver with N-nitrosodiethylamine (DEN)-induced carcinogenesis: these were 26 and 24 kDa species. Here we show that a 2 kDa C-terminal fragment was cleaved from the 26 kDa species in vitro by thrombin or microsomal fractions isolated from DEN-treated rat livers. When Ser(204) of the 26 kDa protein was replaced with Ala by site-directed mutagenesis, phosphorylation of the protein was completely abolished, indicating Ser(204) to be the site of phosphorylation. We also show that the phosphorylation was performed by Ca(2+)-independent protein kinase isoenzymes, and that the phosphorylated rat MGMT protein was resistant to digestion by protease(s) whose activity was increased during DEN-induced hepatocarcinogenesis and also by digestion with endopeptidase Glu-C (V8 protease).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Blotting, Western
  • Calcium / physiology
  • Carcinogens / pharmacology
  • Diethylnitrosamine / pharmacology
  • Endopeptidases / metabolism*
  • Enzyme Induction / drug effects
  • Humans
  • Isoenzymes / metabolism
  • Liver Neoplasms / chemically induced
  • Liver Neoplasms / enzymology
  • Liver Neoplasms / metabolism
  • Male
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • O(6)-Methylguanine-DNA Methyltransferase / chemistry*
  • O(6)-Methylguanine-DNA Methyltransferase / genetics
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism*
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism*
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Sequence Alignment
  • Serine Endopeptidases / metabolism
  • Thrombin / metabolism

Substances

  • Carcinogens
  • Isoenzymes
  • Phosphoserine
  • Diethylnitrosamine
  • O(6)-Methylguanine-DNA Methyltransferase
  • Protein Kinase C
  • Endopeptidases
  • Serine Endopeptidases
  • glutamyl endopeptidase
  • Thrombin
  • Calcium