DNA-dependent protein kinase in human cells

University dissertation from Annahita Sallmyr, Dept of Medical Microbiology, Malmö University Hospital, Entrance 78, 5th floor, SE-205 02 Malmö, SWEDEN

Abstract: DNA-dependent protein kinase (DNA-PK) is a holoenzyme consisting of a regulatory subunit, the heterodimeric Ku70/86, and a catalytic subunit known as DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK takes part in a number of cellular functions including growth control, immunoglobulin gene rearrangement and DNA repair. The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK, causing a dominant-negative inhibition of DNA repair. In the present work we demonstrate that B lymphocytes show apparently full length Ku86 and display DNA-dependent kinase activity. However, a minor fraction of Ku86 in lymphocytes was observed to be truncated. The amount of variant Ku86 is strongly increased in human peripheral blood mononuclear cells (PBMC) by storage of blood prior to the isolation of PBMC. We report that formation of variant Ku86 in protein extracts is mediated by an inducible trypsin-like serine protease with a higher concentration in the nuclear compartment, as compared with the cytoplasm. However, whole cell analysis yielded no evidence of truncated Ku86, suggesting that the protease is not active in intact cells, but is exerting a marked activity during the protein extraction procedure. Interestingly, the protease level became markedly reduced upon transfer of the cells to growth medium. Protease induction did not correlate with apoptosis, necrotic cell death or with signs of general proteolysis or cytotoxicity. Human polymorphonuclear leucocytes (PMN) have been reported to completely lack DNA-PK, and promyelocytic HL-60 cells to express a variant form of Ku resulting in enhanced radiation sensitivity. Here we confirmed the complete lack of DNA-PK in PMN protein extracts, and the expression of the truncated Ku86 variant form in HL-60 extracts. However, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, although at a much reduced level, as compared with HL-60. Our findings have methodological implications for the interpretation of experimental Ku86 data, and suggest that this protease may play a role for cellular regulation of Ku function. To examine the stress response, including the role of DNA-PK in patients with autoimmune disease, B-cell lines were exposed to gamma-radiation and then post-incubated to allow for inducible stress functions to develop. An enhanced DNA damage response could be demonstrated, with respect to DNA-PK phosphorylation of a p53 peptide, flow cytometry analysis of cell cycle phases and apoptosis. These data are in agreement with previous results from studies on Sjögren’s syndrome, suggesting an elevated stress response in these patients.

  This dissertation MIGHT be available in PDF-format. Check this page to see if it is available for download.