PROTEOLYTIC ACTIVATION OF PROTEIN-KINASE-C-DELTA BY AN ICE-LIKE PROTEASE IN APOPTOTIC CELLS

These studies demonstrate that treatment of human U-937 cells with ion izing radiation (IR) is associated with activation of a cytoplasmic my elin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa pr otein, Substrate and inhibitor studies further support the induction o f protein kinase C (PKC)-like activity, The results of N-terminal amin o acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was u sed to confirm proteolytic cleavage of intact 78 kDa PKC delta in cont rol cells to the 40 kDa C-terminal fragment after IR exposure, The fin ding that both IR-induced proteolytic activation of PKC delta and endo nucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-x(L) suppor ts an association with physiological cell death (PCD). Moreover, cleav age of PKC delta occurs adjacent to aspartic acid at a site (QDN) simi lar to that involved in proteolytic activation of interleukin-1 beta c onverting enzyme (ICE), The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate tha t PCD is associated with proteolytic activation of PKC delta by an ICE -like protease.