c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment.
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.
Clem
R J
RJ
Department of Molecular Microbiology and Immunology, Johns Hopkins Schools of Public Health and Medicine, Baltimore, Maryland 21205, USA.
Sheu
T T
TT
Richter
B W
BW
He
W W
WW
Thornberry
N A
NA
Duckett
C S
CS
Hardwick
J M
JM
D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D016466:CHO Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D017353:Gene Deletion; D006801:Humans; D015151:Immunoblotting; D051034:Inhibitor of Apoptosis Proteins; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012845:Sindbis Virus / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D051034:Inhibitor of Apoptosis Proteins; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D015032:Zinc