Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment.
Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.
Ueffing
N
N
Institute of Molecular Medicine, University of Düsseldorf, Universitätsstrasse 1, Düsseldorf D-40225, Germany.
Keil
E
E
Freund
C
C
Kühne
R
R
Schulze-Osthoff
K
K
Schmitz
I
I
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D021621:Imaging, Three-Dimensional; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506387:Cflar protein, mouse; D053418:Death Domain Receptor Signaling Adaptor Proteins; D020033:Protein Isoforms; D014764:Viral Proteins; D053181:Caspase 8; D046988:Proteasome Endopeptidase Complex