Activation of DNA-PK by ionizing radiation is mediated by protein phosphatase 6

PLoS One. 2009;4(2):e4395. doi: 10.1371/journal.pone.0004395. Epub 2009 Feb 9.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Nucleus / enzymology
  • Cell Nucleus / metabolism
  • DNA Damage
  • DNA Repair
  • DNA-Activated Protein Kinase / genetics
  • DNA-Activated Protein Kinase / metabolism*
  • Humans
  • Microscopy, Fluorescence
  • Phosphoprotein Phosphatases / analysis
  • Phosphoprotein Phosphatases / metabolism*
  • Protein Subunits / analysis
  • Protein Subunits / metabolism
  • RNA, Small Interfering / metabolism
  • Radiation, Ionizing*

Substances

  • Protein Subunits
  • RNA, Small Interfering
  • DNA-Activated Protein Kinase
  • Phosphoprotein Phosphatases
  • protein phosphatase 6