Protein phosphorylation is a regulatory mechanism for O-6-alkylguanine-DNAalkyltransferase in human brain tumor cells

The biochemical regulation of human O-6-alkylguanine-DNA alkyltransferase ( AGT), which determines the susceptibility of normal tissues to methylating carcinogens and resistance of tumor cells to many alkylating agents, is poo rly understood. We investigated the regulation of AGT by protein phosphoryl ation in a human medulloblastoma cell line. Incubation of cell extracts wit h [gamma-P-32]ATP resulted in Mg2+-dependent phosphorylation of the endogen ous AGT. Immunoprecipitation after exposure of the cells to P-32-labeled in organic phosphate showed that AGT exists as a phosphoprotein under physiolo gical conditions. Western analysis and chemical stability studies showed th e AGT protein to be phosphorylated at tyrosine, threonine. and serine resid ues. Purified protein kinase A (PKA), casein kinase II (CK II), and protein kinase C (PKC) phosphorylated the recombinant AGT protein with a stoichiom etry of 0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein), resp ectively. Residual phosphorylation of the endogenous AGT by the PKs present in cell homogenates and phosphorylation of the recombinant AGT by purified serine/threonine kinases. PKA, PKC, and CK II reduced AGT activity by 30-6 5%. Conversely, dephosphorylation of cell extracts by alkaline phosphatases stimulated AGT activity. We also identified consensus phosphorylation moti fs for many cellular kinases, including PKA and CK II in the AGT protein. T hese data provide the first and conclusive evidence of AGT phosphorylation and suggest that reversible phosphorylation may control the activity of thi s therapeutically important DNA repair protein in human normal and cancer c ells.