TY - JOUR
T1 - Activation-induced aggregation and processing of the human fas antigen
T2 - Detection with cytoplasmic domain-specific antibodies
AU - Kamitani, Tetsu
AU - Nguyen, Hung Phi
AU - Yeh, Edward T.H.
PY - 1997/8/29
Y1 - 1997/8/29
N2 - 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 β-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.
AB - 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 β-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.
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U2 - 10.1074/jbc.272.35.22307
DO - 10.1074/jbc.272.35.22307
M3 - Article
C2 - 9268381
AN - SCOPUS:0030808570
SN - 0021-9258
VL - 272
SP - 22307
EP - 22314
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 35
ER -