c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

J Biol Chem. 2001 Mar 9;276(10):7602-8. doi: 10.1074/jbc.M010259200. Epub 2000 Dec 5.

Abstract

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis*
  • Binding Sites
  • CHO Cells
  • Caspase 3
  • Caspases / metabolism*
  • Cell Line
  • Cricetinae
  • Gene Deletion
  • Humans
  • Immunoblotting
  • Inhibitor of Apoptosis Proteins
  • Models, Genetic
  • Mutagenesis, Site-Directed
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Sindbis Virus / genetics
  • Transfection
  • Viral Proteins / chemistry*
  • Viral Proteins / metabolism*
  • Zinc / metabolism

Substances

  • Inhibitor of Apoptosis Proteins
  • Viral Proteins
  • inhibitor of apoptosis, Nucleopolyhedrovirus
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Zinc