INTERLEUKIN-1-BETA-CONVERTING ENZYME-LIKE PROTEASE CLEAVES DNA-DEPENDENT PROTEIN-KINASE IN CYTOTOXIC T-CELL KILLING

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming pr otein, protein, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CT L-mediated cytolysis the catalytic subunit of DNA-dependent protein ki nase (DNA-PKcs), an enzyme implicated in the repair of double strand b reaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-co nverting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4- dichloroisocoumarin (DCI), which is known to block granzyme B activity , inhibited CTL-induced apoptosis and prevented the degradation of DNA -PKcs in cells but filled to prevent the degradation of purified DNA-P Kcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and oth er cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with gra nzyme B did not produce the same cleavage pattern observed in cells un dergoing apoptosis and when this substrate was incubated with either C TL extracts or the ICE-like protease, CPP32. Sequence analysis reveale d that the cleavage site in DNA-PKcs during CTL killing was the same a s that when this substrate was exposed to CPP32. This study demonstrat es for the first time that the cleavage of DNA-PKcs in this intact cel l system is exclusively due to an ICE-like protease.