Purification and identification of a protein kinase activity modulated by ionizing radiation.
In order to identify novel protein kinases, which are involved in signal transduction processes after exposure of cells to ionizing radiation we screened HL-60 cells using an in-gel renaturation assay. Using this approach we identified a renaturable serine/threonine kinase with an apparent molecular mass of 90 kDa (pK90). The activity of pK90 dropped within minutes after exposure to a dose of 10 Gy. It reached a minimum 15-30 min after irradiation and increased back to pre-treatment values 6h later. A down-regulation of the kinase activity was detectable after a dose of 1 Gy. Failure of H(2)O(2) to down-regulate pK90 activity indicates a requirement for DNA double-strand-breaks to modulate the kinase activity. In contrast to the molecular mass of 90 kDa in SDS-PAGE we found a molecular mass of around 450 kDa for the native protein using gel filtration chromatography, indicating that pK90 forms a multi-protein complex under native conditions. To identify pK90 we partially purified the protein by three affinity chromatography steps (heparin-Sepharose, phosphate metal affinity, and Cibacron-Blue-F3G-A-Sepharose). Mass spectrometric analysis of the purified 90 kDa fraction showed that pK90 is identical to Tlk1, which was verified by immunoprecipitation.
Kodym
Reinhard
R
Department of Radiobiology, Clinic for Radiotherapy and Radiobiology, University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna, Austria. kodyrel@akh-wien.ac.at
Mayerhofer
Thomas
T
Ortmann
Elisabeth
E
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018922:HL-60 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation; D011233:Precipitin Tests; D020673:Protein Renaturation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000528:radiation effects; D011839:Radiation, Ionizing; D013347:Subcellular Fractions / Q000378:metabolism
D019606:Phosphoamino Acids; D017346:Protein Serine-Threonine Kinases; C425323:TLK1 protein, human