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...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2004-01, Vol.313 (1), p.97-103
Main Authors: Kodym, Reinhard, Mayerhofer, Thomas, Ortmann, Elisabeth
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chromatography
Chromatography, Affinity
Chromatography, Gel
Down-Regulation
Electrophoresis, Polyacrylamide Gel - methods
HL-60 Cells
Humans
Ionizing radiation
Mass Spectrometry - methods
Molecular Weight
Phosphoamino Acids - analysis
Phosphorylation
Precipitin Tests
Protein kinase
Protein Renaturation
Protein-Serine-Threonine Kinases - chemistry
Protein-Serine-Threonine Kinases - isolation & purification
Protein-Serine-Threonine Kinases - metabolism
Protein-Serine-Threonine Kinases - radiation effects
Radiation, Ionizing
Renaturation
Subcellular Fractions - metabolism
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Summary: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.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2003.11.090