Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate, FLICE-like inhibitory protein (FLIP).
We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSSLLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p43) and a C-terminal one (p12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD(376)GPAMKNVEF, identified on N-terminal sequencing of the p12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblotting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-gamma-treated U937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U937 cells treated with vitamin D(3) or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U937 differentiation occur upstream of caspase-8 processing/activation.
Niikura
Yuichi
Y
Division of Cell Biology and Biochemistry, Department of Basic Medical Science, Institute of Medical Science, The University of Tokyo (IMSUT), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Nonaka
Takashi
T
Imajoh-Ohmi
Shinobu
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000276:immunology / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D006887:Hydroxycholecalciferols / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D014212:Tretinoin / Q000494:pharmacology; D020298:U937 Cells; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D006887:Hydroxycholecalciferols; D047908:Intracellular Signaling Peptides and Proteins; D019014:fas Receptor; D014212:Tretinoin; D007371:Interferon-gamma; D020169:Caspases