PROTEASE INVOLVEMENT IN FODRIN CLEAVAGE AND PHOSPHATIDYLSERINE EXPOSURE IN APOPTOSIS

A detailed kinetic analysis of three extranuclear end points of apopto sis, phosphatidylserine exposure, alpha-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentat ion and the activation of the intertleukin-1 beta-converting enzyme (I CE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas mono clonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine e xposure was quantitated by plasma clotting time, as well as annexin V- fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substr ate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited I CE-like activity in both cell types studied, whereas the calpain inhib itor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurka t cells stimulated by anti-Fas monoclonal antibody, indicating that IC E-like proteases play an important role in the regulation of this apop totic system, Calpain inhibitors were ineffective in this system. TNF- induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Ju rkat cells. This suggests that ICE-like enzymes predominate in anti-Fa s monoclonal antibody-stimulated Jurkat cells, whereas proteases affec ted by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells, Impor tantly, the two apoptotic systems seem to be regulated by different pr oteases.