Improved measurement of tyrosine-phosphorylated DNA-PKcs by isoelectric focusing as an indirect determination of DNA-PK activity in vivo.
Isolation of various phosphorylated forms of DNA-PKcs by SDS gel electrophoresis is difficult because of the same molecular weight. By isoelectric focusing (IEF), we confirmed a de-phosphorylated (pI 9.0) and a phosphorylated (pI 6.9) form of DNA-PKcs. When cells were damaged by X-irradiation (5 Gy), an additional tyrosine-phosphorylated (p-Tyr) DNA-PKcs form (pI 6.3) appeared in both nuclear and cytoplasmic fractions, after 30 minsutes.


CONCLUSIONS
IEF in combination with Western blotting of p-Tyr-DNA-PKcs results in an appropriate separation of the p-Tyr form from other phosphorylated forms of DNA-PKcs, which may permit an indirect measurement of changes in DNA-PK activity in irradiated cells.
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Department of Oncology, KFC, Huddinge University Hospital, Stockholm, Sweden. Qimin.He@onkpat.ki.se
Welander
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D000818:Animals; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D007526:Isoelectric Point; D008297:Male; D051379:Mice; D009687:Nuclear Proteins; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012513:Sarcoma, Experimental / Q000201:enzymology
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases