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
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution / genetics
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Animals
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Blotting, Western
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Calcium / physiology
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Carcinogens / pharmacology
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Diethylnitrosamine / pharmacology
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Endopeptidases / metabolism*
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Enzyme Induction / drug effects
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Humans
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Isoenzymes / metabolism
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Liver Neoplasms / chemically induced
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Liver Neoplasms / enzymology
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Liver Neoplasms / metabolism
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Male
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Microsomes, Liver / drug effects
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Microsomes, Liver / enzymology
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Microsomes, Liver / metabolism
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Molecular Sequence Data
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Molecular Weight
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O(6)-Methylguanine-DNA Methyltransferase / chemistry*
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O(6)-Methylguanine-DNA Methyltransferase / genetics
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O(6)-Methylguanine-DNA Methyltransferase / metabolism*
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Phosphorylation / drug effects
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Phosphoserine / metabolism*
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism
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Rats
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Rats, Sprague-Dawley
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Sequence Alignment
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Serine Endopeptidases / metabolism
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Thrombin / metabolism
Substances
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Carcinogens
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Isoenzymes
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Phosphoserine
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Diethylnitrosamine
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O(6)-Methylguanine-DNA Methyltransferase
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Protein Kinase C
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Endopeptidases
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Serine Endopeptidases
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glutamyl endopeptidase
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Thrombin
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Calcium