The Inhibitor of Apoptosis Protein-Binding Domain of Smac Is Not Essential for Its Proapoptotic Activity

doi:10.1083/jcb.153.1.221

Published online 2 April 2001. doi:10.1083/jcb.153.1.221

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© The Rockefeller University Press, 0021-9525/2001//221 $5.00
The Journal of Cell Biology, Volume 153, Number 1, , 2001 221-228

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The Inhibitor of Apoptosis Protein-Binding Domain of Smac Is Not Essential for Its Proapoptotic Activity

Darren L. Roberts^a, Wendy Merrison^a, Marion MacFarlane^a, and Gerald M. Cohen^a

^a Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom
MRC Toxicology Unit, Hodgkin Bldg., University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.44-116-252-561644-116-252-5553

mm21{at}le.ac.uk

Smac/DIABLO, a recently identified inhibitor of apoptosis protein(IAP)-binding protein, is released from the mitochondria duringapoptosis and reportedly potentiates apoptosis by relievingthe inhibition of IAPs on caspases. We now describe the molecularcharacterization of Smac β, an alternatively spliced formof Smac, which lacks the mitochondrial-targeting sequence foundin Smac and has a cortical distribution in both human embryonickidney 293 and breast epithelial tumor MCF-7 cells. Smac β,which binds IAPs in vitro, does not bind IAPs in intact cellsdue to cellular processing and removal of its NH₂-terminal IAP-bindingdomain. Despite its inability to interact with IAPs in cells,processed Smac β is proapoptotic, as demonstrated by itsability to potentiate apoptosis induced by both death receptorand chemical stimuli. Furthermore, expression of a NH₂-terminallytruncated Smac mutant ( ${Delta}$ 75), which lacks the entire IAP-interactingdomain, potentiates apoptosis to the same extent as Smac andSmac β. Our data support the hypothesis that the main proapoptoticfunction of Smac and Smac β is due to a mechanism otherthan IAP binding.

Key Words: TRAIL • XIAP • alternative splicing • MCF-7 cells • mitochondria

Abbreviations used in this paper: HSV, herpes simplex virus; IAP, inhibitor of apoptosis protein; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal;TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducingligand; XIAP, X-linked IAP.

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