Abstract
Caspase-3 and -7 represent executioner/effector caspases that directly cause apoptotic morphological changes by cleaving various death substrates. The substrates for caspases generally interact with active caspases, but not with inactive zymogens of caspase or procaspases. Here, to isolate proteins that interact with caspase-7, we established a yeast two-hybrid screening system using reversed-caspase-7, a constitutive active mutant of caspase-7 as a bait plasmid. Screening of an adult brain cDNA library led to isolation of proteasome activator 28 subunit, PA28γ. In vitro translates of PA28γ were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28γ was cleaved during not only Fas-induced apoptosis of HeLa cells, but also cisplatin-induced cell death of MCF7 cells, which are devoid of caspase-3. These findings indicate that PA28γ is an endogenous substrate for caspase-3 and -7 and that yeast two-hybrid screening using reversed-caspase is a novel and useful approach to clone substrates for effector caspases.
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Abbreviations
- rev-caspase:
-
reversed caspase
- rev-caspase-7-C/S:
-
the active site mutant of reversed caspase-7
- PA28γ:
-
proteasome activator 28 subunit 3
- XIAP:
-
X-linked inhibitor of apoptosis protein
- zVAD-fmk:
-
benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone
- zDEVD-fmk:
-
benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone
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Acknowledgements
We thank Dr. John Reed, Dr. Guy Salvesen and Dr. Paul Friesen for cDNAs for IAPs, caspases and p35, respectively. We also thank Dr. Yasuyuki Suzuki and Dr. Yuzuru Imai for valuable advice and discussion. This work was funded by research grants from RIKEN BSI, a Grant-in-Aid from the Japan Society for the Promotion of Science and grants from the Ministry of Health and Welfare, Japan.
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Araya, R., Takahashi, R. & Nomura, Y. Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28γ as an effector caspase substrate. Cell Death Differ 9, 322–328 (2002). https://doi.org/10.1038/sj.cdd.4400949
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DOI: https://doi.org/10.1038/sj.cdd.4400949
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