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Papers In Press, published online ahead of print March 16, 2001
J. Biol. Chem, 10.1074/jbc.M011703200
Submitted on December 26, 2000
Revised on March 12, 2001
Accepted on March 15, 2001
Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4
Corresponding Author: leesmill{at}ucalgary.ca
DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA-end binding Ku 70/Ku 80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of Non Homologous End Joining (NHEJ). NHEJ is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation, and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku 70 and Ku 80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation induced loss of the protein kinase activity of DNA-PK is restored by the addition of purified catalytic subunit of either protein phosphatase 1 (PP1), or protein phosphatase 2A (PP2A), and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, caused a 50-60% decrease in DNA-PK protein kinase activity, even though the PP1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70 and Ku80 was observed when cells were labeled with 32P-inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity, and that the protein phosphatase responsible for re-activation in vivo is a PP2A-like enzyme.
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