Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.
The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.
*Departments of Biochemistry & Molecular Biology and Oncology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada, T2N 4N1.
Pauline
P
Douglas
*Departments of Biochemistry & Molecular Biology and Oncology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada, T2N 4N1.
Ruiqiong
R
Ye
†Department of Cellular & Molecular Medicine and Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5.
Laura
L
Trinkle-Mulcahy
‡Departments of Pathobiology & Diagnostic Investigation, and Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan, U.S.A.
Jessica A
JA
Neal
§Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 4N1.
Veerle
V
De Wever
∥Beatson Institute for Cancer Research, Glasgow G61 1BD Scotland, U.K.
Nick A
NA
Morrice
‡Departments of Pathobiology & Diagnostic Investigation, and Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan, U.S.A.
Katheryn
K
Meek
*Departments of Biochemistry & Molecular Biology and Oncology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada, T2N 4N1.
Susan P
SP
Lees-Miller
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D051747:DNA-Activated Protein Kinase; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1; C578634:CHEK2 protein, human; C497263:PRKDC protein, human; C106079:protein phosphatase 6