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 6 h later. A down-regulation of the kinase activity was detectable after a dose of 1 Gy. Failure of H 2O 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.
Published Version
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