The C. elegans orthologue ceBNIP3 interacts with CED-9 and CED-3 but kills through a BH3- and caspase-independent mechanism.
We have studied ceBNIP3, the orthologue of BNIP3 in C. elegans. Sequence analysis reveals that the different domains of BNIP3 have been conserved throughout evolution. ceBNIP3 contains a C-terminal transmembrane (TM) domain, a conserved domain (CD) of 19 amino acids, a BCL-2 homology-3 (BH3)-like domain and a PEST sequence. ceBNIP3 is expressed primarily as a 25 kDa monomer and a 50 kDa homodimer. After transfection, ceBNIP3 protein is rapidly degraded through a ubiquitin-dependent pathway by the proteasome. Like BNIP3, the TM domain of ceBNIP3 mediates the localization of the protein to mitochondria and is also necessary for homodimerization and cell death in mammalian cells. Neither the putative BH3 domain nor conserved domain is necessary for killing. ceBNIP3 protein interacts with CED-9 and BCL-XL, but unlike other pro-apoptotic BCL-2 family members, the BH3-like domain does not participate in dimerization. The ceBNIP3 TM domain mediates interaction with both CED-9 and BCL-XL. ceBNIP3 interacts with CED-3 but co-expression of CED-3 and ceBNIP3 does not significantly enhance induction of cell death in the presence or absence of CED-4. ceBNIP3 kills mammalian cells by a caspase-independent mechanism. In conclusion, we find that although ceBNIP3 interacts with CED-9 and CED-3 it kills by a BH3- and caspase-independent mechanism.
Cizeau
J
J
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
Ray
R
R
Chen
G
G
Gietz
R D
RD
Greenberg
A H
AH
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology*; D002460:Cell Line; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019281:Dimerization; D005786:Gene Expression Regulation; D015801:Helminth Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; C494811:BCL2L1 protein, human; C116838:BNIP3L protein, human; C494812:Bcl2l1 protein, mouse; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C087233:Ced-9 protein, C elegans; D015853:Cysteine Proteinase Inhibitors; C115798:DCT-1 protein, C elegans; D015801:Helminth Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases; D003546:Cysteine Endopeptidases; C089223:ced-3 protein, C elegans