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Protein Phosphorylation Is a Regulatory Mechanism for O⁶-Alkylguanine-DNA Alkyltransferase in Human Brain Tumor Cells¹

Section of Molecular Therapeutics, Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [K. S. S., S. R. S. M., J. S., F. A.-O.], and the Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555 [T. K. H.]

The biochemical regulation of human O⁶-alkylguanine-DNA alkyltransferase (AGT), which determines the susceptibility of normal tissues to methylating carcinogens and resistance of tumor cells to many alkylating agents, is poorly understood. We investigated the regulation of AGT by protein phosphorylation in a human medulloblastoma cell line. Incubation of cell extracts with [-³²P]ATP resulted in Mg²⁺-dependent phosphorylation of the endogenous AGT. Immunoprecipitation after exposure of the cells to ³²P-labeled inorganic phosphate showed that AGT exists as a phosphoprotein under physiological conditions. Western analysis and chemical stability studies showed the AGT protein to be phosphorylated at tyrosine, threonine, and serine residues. Purified protein kinase A (PKA), casein kinase II (CK II), and protein kinase C (PKC) phosphorylated the recombinant AGT protein with a stoichiometry of 0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein), respectively. 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 30–65%. Conversely, dephosphorylation of cell extracts by alkaline phosphatases stimulated AGT activity. We also identified consensus phosphorylation motifs for many cellular kinases, including PKA and CK II in the AGT protein. These data provide the first and conclusive evidence of AGT phosphorylation and suggest that reversible phosphorylation may control the activity of this therapeutically important DNA repair protein in human normal and cancer cells.

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