The catalytic subuntt of dna-dependent protein kinase is cleaved during fas-mediated apoptosis in jurkat cells

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase linked to DNA repair, cell cycle progression and V(D)J recombination. It is composed of an approximately 460 kDa catalytic subunit (DNA-PK_cs) and the human autoantigen Ku (p70 and p86), which serves to target the catalytic subunit to the free ends of double-stranded DNA breaks. Since apoptosis is associated with formation of dsDNA breaks, we examined in Jurkat cells the behavior of the catalytic subunit during Fas (Apo-1, CD95) mediated apoptosis. Using sera from patients with connective tissue disease, polyclonal antibodies or a series of monoclonal antibodies directed toward DNA-PKcs, we found by immunoprecipitation of metabolically-labeled lysates that the catalytic subunit was cleaved into several smaller polypeptides at the onset of apoptosis. Some polypeptides appeared to remain associated with the Ku subunit after cleavage. Additional polypeptide fragments appeared as apoptosis progressed. Western blotting confirmed that the smaller polypeptides arose from DNA-PK_cs and were not merely proteins which became associated with DNA-PK_cs during apoptosis. In contrast to DNA-PK_cs, the Ku subunits were neither cleaved nor decreased in amount during apoptosis, nor were other common autoantigens cleaved during the time required for DNA-PKcs cleavage. Finally, resting lymphocytes were more resistant than cycling lymphocytes to Fas-mediated apoptosis. We surmise that cleavage of DNA-PK_cs may represent a mechanism for regulating the function of DNA-PK during programmed cell death. Whether the polypeptides which result from apoptosis-mediated cleavage of DNA-PK_cS incite an autoimmune response remains to be determined.