D4-GDI, A SUBSTRATE OF CPP32, IS PROTEOLYZED DURING FAS-INDUCED APOPTOSIS

Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulat ion of the immune system, normal morphogenesis, and maintenance of hom eostasis. ICE/CED-3 family cysteine proteases have been implicated dir ectly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4 -GDI, an abundant hematopoietic cell GDP dissociation inhibitor for th e Ras-related Rho family GTPases, is a substrate of the apoptosis prot ease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa frag ment in Jurkat cells with kinetics that parallel the onset of apoptosi s following Fas cross-linking with agonistic antibody or treatment wit h staurosporine. Fas- and staurosporine-induced apoptosis as well as c leavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GD I was cleaved in vitro by recombinant CPP32 expressed in Escherichia c oil to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI w as completely inhibited by 1 mu M DEVD-CHO, a reported selective inhib itor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrat ions up to 50 mu M. N-terminal sequencing of the 23-kDa D4-GDI fragmen t demonstrated that D4-GDI was cleaved between Asp(19) and Ser(20) of the poly(ADP-ribose) polymerase-like cleavage sequence DELD(19)S. Thes e data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI prote in.