The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its antiapoptotic effect.A poptosis is a physiological cell suicide program critical to the development and homeostasis of all animals (1). Abnormal inhibition of apoptosis is a hallmark of cancer and autoimmune disease, whereas excessive cell death has been implicated in neurodegenerative disorders (2). The caspases, a family of intracellular cysteine proteases, are the central executioners of apoptosis (3). Effector caspases, such as caspase-3 and -7, are activated by initiator caspases, such as caspase-9, through proteolytic cleavage (3). Once activated, the effector caspases are responsible for the proteolytic cleavage of a diverse array of structural and regulatory proteins, resulting in an apoptotic phenotype (4).Inhibitor of apoptosis proteins (IAPs), originally found in baculoviruses, have been conserved in a number of species, ranging from insects to humans throughout evolution and play a role in regulating apoptosis (5, 6). Several members of the human IAP family proteins, including X-linked IAP (XIAP), c-IAP1 and c-IAP2, have been shown to be potent direct inhibitors of caspase-3, -7, and -9 (7-9). Among the above IAPs, XIAP is the most potent inhibitor of caspases and apoptosis (6). The structure of XIAP is characterized by three tandem repeats of the baculovirus IAP repeat (BIR) domain at its NH 2 terminus and a RING finger domain near its COOH terminus. Deletional analysis indicates that the second BIR domain (BIR2) of XIAP is sufficient to inhibit caspase-3 and -7 (10), whereas a XIAP fragment encompassing the third BIR domain (BIR3) and the RING finger domain specifically inhibits caspase-9 (11). A recent report indicates that only the BIR3 domain of XIAP is required to inh...

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH 2 -terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH 2 -terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.Inhibitor of apoptosis proteins (IAPs) 1 , originally found in baculoviruses, play evolutionary conserved roles from insects to humans in regulating programmed cell death and apoptosis (1, 2). Several members of human IAP family proteins including X-linked IAP (XIAP), c-IAP1, and c-IAP2 have been shown to be potent direct inhibitors of a subset of cell death proteases, caspase-3, -7 and -9 (3-5). Among the above caspase-inhibiting IAPs, XIAP has the most potent anti-apoptotic effect in cells with the lowest K i for purified caspase-3 and -7 in vitro (3). The structure of XIAP is characterized by three tandem repeats of 70 amino acids motif called baculovirus IAP repeat (BIR) domain at its NH 2 terminus and a RING zinc finger domain near its COOH terminus. Structure-function analysis of XIAP has shown that the region of XIAP containing the second BIR domain is necessary and sufficient for inhibiting caspase-3 and -7 (6), whereas the third BIR domain is responsible for the inhibition of caspase-9 (7,8). A recent report of the NMR solution-structure of XIAP-BIR2 domain revealed that it is similar to classical zinc finger motifs, consisting of a threestranded antiparallel ␤-sheet and four ␣-helices (9). Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were shown to be essential for inhibiting caspase-3 (9). Moreover it was suggested that these residues might bind to the active site, whereas the BIR domain might interact with an adjacent site on caspase-3 (9). However, the detailed molecular mechanism of XIAP-mediated inhibition of caspase-7, in comparison with caspase-3, has yet to be shown.Here, we showed that the inhibitory mode of c...