Phosphorylation of methylated-DNA-protein-cysteine S-methyltransferase at serine-204 significantly increases its resistance to proteolytic digestion.
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).
Lim
I K
IK
Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 442-749, Korea. iklim@madang.ajou.ac.kr
Park
T J
TJ
Paik
W K
WK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002273:Carcinogens / Q000494:pharmacology; D004052:Diethylnitrosamine / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008113:Liver Neoplasms / Q000139:chemically induced / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D002273:Carcinogens; D007527:Isoenzymes; D010768:Phosphoserine; D004052:Diethylnitrosamine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011493:Protein Kinase C; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013917:Thrombin; D002118:Calcium