PHOSPHATIDYLSERINE EXTERNALIZATION DURING CD95-INDUCED APOPTOSIS OF CELLS AND CYTOPLASTS REQUIRES ICE CED-3 PROTEASE ACTIVITY/

Phosphatidylserine (PS), a lipid normally confined to the inner leafle t of the plasma membrane, is exported to the outer plasma membrane lea flet during apoptosis to serve as a trigger for recognition of apoptot ic cells by phagocytes. The mechanism of PS export during apoptosis is not known nor is it clear whether the nuclear changes that typify apo ptosis contribute in any way to this event. Here, we demonstrate that ligation of the CD95 (Fas/APO-1) molecule on Jurkat cytoplasts induces dramatic PS externalization similar to that observed during apoptosis of intact cells. Apoptosis of both cells and cytoplasts was associate d with proteolytic processing of CPP32, a member of the interleukin-1 beta converting enzyme (ICE)/CED-3 protease family, to its active form . Fodrin, a component of the cortical cytoskeleton, also underwent pro teolytic cleavage during apoptosis of both cytoplasts and intact cells . Strikingly, CPP32 activation, fodrin proteolysis, and PS externaliza tion were all inhibited in the presence of peptide inhibitors of ICE/C ED-3 family proteases. These data provide strong support for the notio n that the cell death machinery is extranuclear and is likely to be co mprised of one or more members of the ICE/CED-3 family and that activa tion of this machinery does not require nuclear participation.