Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

Abstract

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 selfoligomerization 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. Death receptors like CD95 (Fas/Apo-1) transduce death signals upon ligand binding and formation of a death-inducing signaling complex (DISC), which comprises the receptor, the adaptor protein Fas-associated death domain (FADD) and procaspase-8 (FLICE) or procaspase-10. 1 This assembly brings procaspase-8/10 in close proximity to the receptor and allows them to be activated by dimerization. 2-4 Activation of the initiator caspases can be counteracted by FLICEinhibitory proteins (FLIPs). 5,6 Next to viral FLIP (v-FLIP) proteins, 7 three cellular homologs (cellular FLICE-inhibitory proteins (c-FLIPs)) have been identified, namely c-FLIP long , c-FLIP short and c-FLIP R , which are generated by differential splicing. [8][9][10] The C-terminus of c-FLIP long contains a catalytically inactive caspase-like domain, whereas c-FLIP short and c-FLIP R have only a truncated C-terminus.As a characteristic feature, FLIP proteins contain tandem death effector domains (DEDs), which mediate their recruitment into the DISC. 10,11 The DED forms a bundle of six antiparallel a-helices similar to the death domain (DD) and the caspase recruitment domain (CARD), two other signaling motifs involved in homotypic protein interactions. 12 Curiously, despite their importance in apoptosis, there are currently no reported structures for cellular FLIP proteins. Only the structure of the tandem DEDs of the v-FLIP MC159 from Molluscum contagiosum has been reported showing a rigidly associated dumbbell-shaped molecule, which is tightly packed by an extensive hydrophobic interface between its two DEDs. 13,14 Similar to FADD, each DED of MC159 contains two prominent surface features important for protein interactions that distinguish them from other death mot...

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

“…The different c-FLIP isoforms are also randomly recruited to the chains via the FL-motif, in a ratio to procaspase-8 of 1-10, which is followed by processing of c-FLIP L to p43-FLIP (Figure 8b). 34,37 Remarkably, our data indicate that the previously suggested mechanism of procaspase-8 inhibition by c-FLIP, based on the competition for binding sites at the DISC, is not valid. Our data indicate that likely c-FLIP S/R has an inhibitory potential, if the DED chain is mostly composed of c-FLIP S/R /procaspase-8 heterodimers, which would prevent procaspase-8 homodimer formation.…”

“…In line with this, it has been reported that the charge triad motif of murine c-FLIP R is not required for its recruitment to the DISC and inhibition of procaspase-8 activation. 37 Chain termination depends on procaspase-8 dissociation/association rates. To strive better understanding of chain termination control, we used mathematical modeling.…”

p63 regulates the caspase-8-FLIP apoptotic pathway in epidermis

“…At the protein level, c-FLIP has three different isoforms -FLIPl, FLIPr and FLIPs -in humans, and two -FLIPl and FLIPr -in mice. [19][20][21][22][23][24] In this report, we demonstrate that p63 activates the genes of both procaspase-8 and of its negative regulator, c-FLIP.…”

Fas death receptor signalling: roles of Bid and XIAP

“…28 Low concentrations of c-FLIP L promote caspase-8 recruitment and activation, whereas high concentrations of c-FLIP L inhibit capase-8 activation, likely due to competition for FADD interaction. 22 Isoforms of c-FLIP lacking its caspase-like domain (c-FLIPs and c-FLIP R , the only short c-FLIP isoform expressed in the mouse, 29 ), as well as viral forms of FLIP act solely as potent inhibitors of caspase-8 recruitment and activation. [29][30][31] The Bcl-2 Protein Family and its Role in the Regulation of Apoptosis…”