DNA-dependent protein kinase in human cells
Research output: Thesis › Doctoral Thesis (compilation)
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.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Award date||2004 Nov 30|
|Publication status||Published - 2004|
Defence details Date: 2004-11-30 Time: 09:15 Place: Main lecture hall, Pathology building, Malmö University Hospital, Entrance 78, 2nd floor External reviewer(s) Name: Lewensohn, Rolf Title: Professor Affiliation: Dept of Medical Radiation Biology, Cancer Center Karolinska R8:00, Karolinska Hospital, 171 76 Stockholm, SWEDEN --- Article: I. Sallmyr, A., Henriksson, G., Fukushima, S., and Bredberg, A. (2001). Ku protein in human T and B lymphocytes: Full length functional form and signs of degradation. Biochimica et Biophysica Acta 1538(2001)305-312 Article: II. Sallmyr, A., Du, L., and Bredberg, A. (2002). An inducible Ku86-degrading serine protease in human cells. Biochimica et Biophysica Acta 1593(2002)57-68 Article: III. Henriksson, G., Sallmyr, A., Du, L., Larsson, Å., Manthorpe, R., and Bredberg, A. (2004). Enhanced DNA-dependent protein kinase activity in Sjögren’s Syndrome B cells. Rheumatology (2004);43:1109-1115 Article: IV. Sallmyr, A., Miller, A., Gabdoulkhakova, A., Safronova, V., Henriksson, G., and Bredberg, A. (2004). Expression of DNA-dependent protein kinase in human granulocytes. Cell Research (2004); 14(4):331-340