Protein phosphorylation is a regulatory mechanism for O6-alkylguanine- DNA alkyltransferase in human brain tumor cells

Kalkunte S. Srivenugopal, Srinivas R.S. Mullapudi, Jiang Shou, Tapas K. Hazra, Francis Ali-Osman

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

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 [γ-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.

Original languageEnglish (US)
Pages (from-to)282-287
Number of pages6
JournalCancer Research
Volume60
Issue number2
StatePublished - Jan 15 2000

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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