Abstract
Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-specific BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a single- vector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-deficient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wild-type HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x(L) and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not sufficient to confer sensitivity to Nbk.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenoviridae / genetics
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Amino Acid Sequence
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Apoptosis Regulatory Proteins
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Apoptosis* / genetics
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Apoptosis* / physiology
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Caspases / metabolism
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Cell Line
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Cytochrome c Group / metabolism
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Gene Expression Regulation, Neoplastic
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Gene Expression Regulation, Viral
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Humans
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Intracellular Membranes / metabolism
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Membrane Proteins / metabolism
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Mitochondria / metabolism*
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Mitochondrial Proteins
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Protein Conformation
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Protein Isoforms
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Proteins / chemistry
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Proteins / genetics
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Proteins / metabolism*
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Proto-Oncogene Proteins / chemistry
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-bcl-2 / chemistry
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Proto-Oncogene Proteins c-bcl-2 / genetics
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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Recombinant Proteins / metabolism
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Tumor Cells, Cultured
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bcl-2 Homologous Antagonist-Killer Protein
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bcl-2-Associated X Protein
Substances
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Apoptosis Regulatory Proteins
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BAK1 protein, human
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BAX protein, human
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BIK protein, human
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Cytochrome c Group
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Membrane Proteins
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Mitochondrial Proteins
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Protein Isoforms
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Proteins
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Recombinant Proteins
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bcl-2 Homologous Antagonist-Killer Protein
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bcl-2-Associated X Protein
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Caspases