Activation of the mitochondrial death pathway is commonly mediated by a preferential engagement of Bak.
Among the members of the Bcl-2 family, the multidomain proteins Bax and Bak are crucial for the activation of mitochondria. However, it is still unclear whether they act in a unique and distinct manner or whether they exhibit redundant functions. To systematically investigate their activation on a single-cell level, we established MCF-7 cell lines stably expressing GFP-fusion variants of these proteins. We found that MCF-7/GFP-Bak cells showed an increased sensitivity to apoptosis induction by staurosporine, actinomycin D, TRAIL and overexpression of Puma compared to GFP-Bax-expressing cells. Independently of the death stimulus used, oligomerization of endogenous and exogenous Bak was mostly detected prior to an activation of Bax, whereas cells displaying oligomerized Bax in the absence of Bak clusters were not observed. In addition, activation of Bax but not Bak was attenuated by a caspase inhibitor. Consistent with this, caspase-3-deficient MCF-7 cells displayed a significantly reduced activation of endogenous Bax than caspase-3-proficient MCF-7 cells. Thus, our data strongly suggest that diverse apoptotic stimuli preferentially engage the Bak pathway, whereas the triggering of Bax occurs, at least partially, downstream of mitochondrial caspase activation, most likely constituting a positive feedback loop for the amplification of the death signal.
Neise
D
D
Institute of Molecular Medicine, University of Düsseldorf, Düsseldorf, Germany.
Graupner
V
V
Gillissen
B F
BF
Daniel
P T
PT
Schulze-Osthoff
K
K
Jänicke
R U
RU
Essmann
F
F
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics; D045744:Cell Line, Tumor; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000502:physiology; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000502:physiology
C494772:BAK1 protein, human; C494826:BAX protein, human; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D053221:TNF-Related Apoptosis-Inducing Ligand; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; D020169:Caspases; D019311:Staurosporine