title: Purification and identification of a protein kinase activity modulated by ionizing radiation.
abstract: 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.
authors.affiliation: Department of Radiobiology, Clinic for Radiotherapy and Radiobiology, University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna, Austria. kodyrel@akh-wien.ac.at
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kodym
authors.forename: Thomas
authors.initials: T
authors.lastname: Mayerhofer
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Ortmann
mesh_terms: 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
chemical_list: D019606:Phosphoamino Acids; D017346:Protein Serine-Threonine Kinases; C425323:TLK1 protein, human