title: Activation of DNA-PK by ionizing radiation is mediated by protein phosphatase 6.
abstract: DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous end-joining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1gamma1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.
authors.affiliation: Department of Radiation Oncology, University of Virginia Health System, Charlottesville, Virginia, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Mi
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Dziegielewski
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Bolesta
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
authors.forename: James M
authors.initials: JM
authors.lastname: Larner
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000378:metabolism*; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011839:Radiation, Ionizing
chemical_list: D021122:Protein Subunits; D034741:RNA, Small Interfering; D051747:DNA-Activated Protein Kinase; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6