Abstract
Caspase-8, a cysteine-protease, initiates apoptosis when activated by death receptors. Caspase-8 is also essential for initiating T lymphocyte proliferation following T-cell antigen receptor (TCR) signaling. Given these disparate functions of caspase-8, we sought to determine whether this represented only a difference in the magnitude of caspase-8 activation, or different intracellular locations of active caspase-8. We demonstrate by high-resolution multicolor confocal laser scanning microscopy an aggregation of active caspase-8 within membrane lipid rafts in T cells stimulated with anti-CD3. This suggests that following TCR stimulation active caspase-8 physically interacts with lipid raft proteins, possibly to form a signaling platform. In contrast, Fas stimulation of T cells resulted in a much more profound activation of caspase-8 that was exclusively cytosolic. These confocal microscopic findings were confirmed using discontinuous sucrose gradient ultracentrifugation to isolate lipid raft versus cytosolic components. This sequestration model of caspase-8 activation was further supported by the observation that a classic caspase-8 substrate, BID, was not cleaved in CD3-stimulated T cells, but was cleaved after Fas engagement. Our data support a model that the location of active caspase-8 may profoundly influence its functional capacity as a regulator of either cell cycling or cell death.
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Abbreviations
- CLSM:
-
confocal laser scanning microscopy
- FAM:
-
6-carboxyfluorescein
- FITC:
-
fluorescein isothiocyanate
- fmk:
-
fluoromethylketone
- IETD:
-
isoleucine-glutamic acid-threonine-aspartic acid
- LETD:
-
leucine-glutamic acid-threonine-aspartic acid
- TCR:
-
T-cell antigen receptor
- TUNEL:
-
terminal-dUTP-nick-end-labeling
- zVAD:
-
benzyloxycarbonyl-valine-alanine-aspartic acid
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Acknowledgements
This work was supported by grants from the National Institutes of Health (AI36333, AI45666 and AR43520 to RCB, and R01GM070001 to WAR). We thank Dr. Douglas Taatjes and Marilyn Wadsworth for help and discussions concerning confocal laser scanning microscopy, and Colette Charland and Julie Wolfe for technical assistance with flow cytometry, and Tim Hunter and Scott Tighe for technical assistance with densitometry.
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Koenig, A., Russell, J., Rodgers, W. et al. Spatial differences in active caspase-8 defines its role in T-cell activation versus cell death. Cell Death Differ 15, 1701–1711 (2008). https://doi.org/10.1038/cdd.2008.100
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DOI: https://doi.org/10.1038/cdd.2008.100
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