Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates gamma-H2AX.
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.
Douglas
Pauline
P
Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.
Zhong
Jianing
J
Ye
Ruiqiong
R
Moorhead
Greg B G
GB
Xu
Xingzhi
X
Lees-Miller
Susan P
SP
D064007:Ataxia Telangiectasia Mutated Proteins; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D016195:G2 Phase / Q000528:radiation effects; D020868:Gene Silencing / Q000528:radiation effects; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008938:Mitosis / Q000528:radiation effects; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011839:Radiation, Ionizing; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000098550:Structural Maintenance of Chromosome Protein 1
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D000098550:Structural Maintenance of Chromosome Protein 1; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6