Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway

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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‐xL 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.