Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies.
Fas (APO1/CD95) is a type 1 transmembrane protein critically involved in receptor-mediated apoptosis. Previous studies have shown that Fas exists in monomeric form in resting cells and aggregates upon cross-linking to form a complex that serves to recruit additional signaling molecules to the cell membrane. To study the molecular fate of the Fas antigen following receptor activation, a monoclonal antibody specific for the cell death domain of Fas has been generated. This monoclonal antibody (3D5) could be used in Western blot analysis using total cell lysates to identify different forms of Fas antigens without immunoprecipitation. High molecular mass (>200 kDa), SDS- and beta-mercaptoethanol-resistant Fas aggregates were formed immediately following receptor cross-linking, and a 97-kDa band (p97) was detected about 2 h later. p97 could be detected by antibodies against either the death domain or the C terminus. However, p97 could not be precipitated by antiextracellular domain antibodies. Thus, p97 most likely represents a processed form of the high molecular weight Fas aggregates. Although p97 generation followed a similar time course as CPP32 activation and poly(ADP-ribose) polymerase cleavage, it could not be inhibited by cysteine protease, calpain, or proteasome inhibitors.
Kamitani
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Department of Internal Medicine, and Cardiovascular Research Center, Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston Health Science Center, Houston, Texas 77030, USA.
Nguyen
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HP
Yeh
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ET
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018604:Epitope Mapping; D005434:Flow Cytometry; D006801:Humans; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000737:chemistry / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D011480:Protease Inhibitors; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases