Abstract
The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G(2)/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (gamma-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of gamma-H2AX, the dissolution of IR-induced foci, and release from the G(2)/M checkpoint in vivo.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Ataxia Telangiectasia Mutated Proteins
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Catalytic Domain*
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Cell Cycle Proteins / metabolism
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Cell Extracts
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Cell Nucleus / metabolism
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Cell Nucleus / radiation effects
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Checkpoint Kinase 2
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Chromosomal Proteins, Non-Histone / metabolism
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Comet Assay
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DNA Breaks, Double-Stranded / radiation effects
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DNA Repair / radiation effects
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DNA-Activated Protein Kinase / metabolism*
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DNA-Binding Proteins / metabolism
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G2 Phase / radiation effects
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Gene Silencing / radiation effects
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HeLa Cells
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Histones / metabolism*
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Humans
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Mitosis / radiation effects
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Models, Biological
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Nuclear Proteins / metabolism*
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Phosphoprotein Phosphatases / metabolism*
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Phosphorylation / radiation effects
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Protein Binding / radiation effects
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Protein Serine-Threonine Kinases / metabolism
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Radiation, Ionizing
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Tumor Suppressor Proteins / metabolism
Substances
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Cell Cycle Proteins
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Cell Extracts
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Chromosomal Proteins, Non-Histone
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DNA-Binding Proteins
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H2AX protein, human
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Histones
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Nuclear Proteins
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Tumor Suppressor Proteins
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structural maintenance of chromosome protein 1
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Checkpoint Kinase 2
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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CHEK2 protein, human
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DNA-Activated Protein Kinase
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PRKDC protein, human
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Protein Serine-Threonine Kinases
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Phosphoprotein Phosphatases
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protein phosphatase 6