Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28gamma as an effector caspase substrate.
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, PA28gamma. In vitro translates of PA28gamma were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28gamma 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 PA28gamma 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.
Araya
R
R
Laboratory for Motor System Neurodegeneration, RIKEN Brain Science Institute, Saitama 351-0198, Japan.
Takahashi
R
R
Nomura
Y
Y
D017209:Apoptosis / Q000502:physiology*; D001324:Autoantigens; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism*; D002945:Cisplatin; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009124:Muscle Proteins; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D013379:Substrate Specificity / Q000502:physiology; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D019014:fas Receptor / Q000378:metabolism
D001324:Autoantigens; C099558:Ki antigen; D009124:Muscle Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D019014:fas Receptor; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; D002945:Cisplatin