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. Cli n. Oncol. 125, 493-499], we showed two major forms of active DNA-6-O-methyl guanine :protein-L-cysteine S-methyltransferase (MGMT; EC 2.1.1.63) in the liver with N-nitrosodiethylamine (DEN)-induced carcinogenesis: these were 2 6 and 24 kDa species. Here we show that a 7 kDa C-terminal fragment was cle aved from the 26 kDa species in vitro by thrombin or microsomal fractions i solated from DEN-treated rat livers. When Ser(204) of the 26 kDa protein wa s replaced with Ala by site-directed mutagenesis, phosphorylation of the pr otein was completely abolished, indicating Ser(204) to be the site of phosp horylation. We also show that the phosphorylation was performed by Ca2+-ind ependent protein kinase isoenzymes, and that the phosphorylated rat MGMT pr otein was resistant to digestion by protease(s) whose activity was increase d during DEN-induced hepatocarcinogenesis and also by digestion with endope ptidase Glu-C (V8 protease).