BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts.
The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, expression of non-apoptotic levels of either ncBID or wtBID in Bid -/- MEFs induced a similar and significant enhancement in apoptosis in response to a variety of death signals, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.
Sarig
Rachel
R
Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
Zaltsman
Yehudit
Y
Marcellus
Richard C
RC
Flavell
Richard
R
Mak
Tak W
TW
Gross
Atan
A
D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000502:physiology; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D009154:Mutation; D011506:Proteins / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D002352:Carrier Proteins; D003574:Cytochrome c Group; D011506:Proteins; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases