Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death

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 anti-apoptotic effect.