Cell cycle-dependent regulation of the DNA-dependent protein kinase.
Human DNA-dependent protein kinase (DNA-PK) is a nuclear-localized serine/threonine protein kinase. The holoenzyme consists of a catalytic subunit with a molecular mass of 465 kDa and a DNA-binding heterodimer Ku86/70. The kinase has been implicated in a variety of nuclear processes including V(D)J recombination, double-strand break repair, and transcription. Cells with defective DNA-PK activity show increased radiosensitivity and lack of V(D)J recombination. To study DNA-PK activity during the cell cycle, HeLa cells were separated by elutriation centrifugation into different cell cycle compartments based on cellular size. DNA-PK activity was found to vary during the cell cycle. The kinase activity was lowest during G1 phase and increased dramatically as the cells entered S phase and remained high during the G2-phase. The subcellular distribution of DNA-PKcs is relocalized from the cytoplasm during M and G1 phases to the nucleus during G1-S phase transition and S phase. Expression of both the catalytic subunit and the Ku86/70 heterodimer was found to be constant throughout the cell cycle. This study demonstrates that DNA-PK activity as well as its subcellular localization fluctuates during the cell cycle. In addition, the distribution of DNA-PK during M phase corresponds with low DNA-PK activity.
Nilsson
A
A
Unit of Medical Radiobiology, Cancer Centre Karolinska, Karolinska Hospital, Stockholm, Sweden. Anders.Nilsson@onkpat.ki.se
Sirzén
F
F
Lewensohn
R
R
Wang
N
N
Skog
S
S
D015153:Blotting, Western; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation / Q000502:physiology; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D016196:S Phase / Q000502:physiology*; D013347:Subcellular Fractions / Q000201:enzymology
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases