Fas-associated protein with death domain (FADD)-independent recruitment of c-FLIPL to death receptor 5

Abstract

Here we show a novel mechanism by which FLICE-like inhibitory protein (c-FLIP) regulates apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and one of its receptors, DR5. c-FLIP is a critical regulator of the TNF family of cytokine receptor signaling. c-FLIP has been postulated to prevent formation of the competent death-inducing signaling complex (DISC) in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. In order to identify regulators of TRAIL function, we used the intracellular death domain (DD) of DR5 as a target to screen a phage-displayed combinatorial peptide library. The DD of DR5 selected from the library a peptide that showed sequence similarity to a stretch of amino acids in the C terminus of c-FLIP(L). The phage-displayed peptide selectively interacted with the DD of DR5 in in vitro binding assays. Similarly, full-length c-FLIP (c-FLIP(L)) and the C-terminal p12 domain of c-FLIP interacted with DR5 both in in vitro pull-down assays and in mammalian cells. This interaction was independent of TRAIL. To the contrary, TRAIL treatment released c-FLIP(L) from DR5, permitting the recruitment of FADD to the active DR5 signaling complex. By employing FADD-deficient Jurkat cells, we demonstrate that DR5 and c-FLIP(L) interact in a FADD-independent manner. Moreover, we show that a cellular membrane permeable version of the peptide corresponding to the DR5 binding domain of c-FLIP induces apoptosis in mammalian cells. Taken together, these findings indicate that c-FLIP interacts with the DD of DR5, thus preventing death (L)signaling by DR5 prior to the formation of an active DISC. Because TRAIL and DR5 are ubiquitously expressed, the interaction of c-FLIP(L) and DR5 indicates a mechanism by which tumor selective apoptosis can be achieved through protecting normal cells from undergoing death receptor-induced apoptosis.

FIG. 1

A, isolated peptide interacts specifically with DR5. Using a phage-displayed combinatorial peptide library, we have identified a peptide that interacts with DR5. Equivalent numbers of phage particles were added to microtiter wells coated with 1 µg each of GST fusion proteins of DR5(DD), DR4(DD), Fas(DD), Src(SH3), Abl (SH3), MP-90, hNOS(PDZ), p53N, E6, or negative controls GST and bovine serum albumin as indicated. The strength of interaction was determined in an ELISA with anti-M13 phage antibody. The isolated peptide specifically interacts with DR5(DD) and only very weakly with the highly homologous relative, DR5(DD) protein. The y-axis is a measure of optical density at 405 nm. B, alanine scanning of the phage peptide reveals the DR5 binding sequence. Equivalent amounts (1 µg/well) of DR5 fusion proteins were immobilized in microtiter plates and incubated with 10¹⁰ phage particles from variants of the isolated phage peptide sequence RGSFWWLETAPP. These variants have one mutation each, sequentially substituting each amino acid in the peptide to alanine. Except for the Glu → Ala substitution, which enhances binding, sequential alanine substitutions of the FWWLET peptide sequence causes loss of binding.

FIG. 2. DR5 and c-FLIP_L interact in vitro

A, interaction of a c-FLIP peptide with DR5 protein. We displayed on the phage a c-FLIP peptide segment resembling the sequence of the peptide ligand selected by the DD of DR5 from a phage-displayed combinatorial peptide library. In this format, the c-FLIP peptide specifically interacts with GST-DR5 protein, but not to unrelated GST fusion proteins, in an ELISA. B, interaction of full-length c-FLIP_L with DR5 in a GST pull-down assay. Cell lysates were prepared from HEK 293 cells ectopically expressing FLAG-tagged c-FLIP_L or FLAG-tagged c-FLIP_p12. These cell lysates were used in a GST pull-down assay with GST fusion proteins of DR5 and immunoblotted with an anti-FLAG antibody. Total cell lysate is used as a control to monitor expression of c-FLIP_L and c-FLIP_p12.

FIG. 3. DR5 and c-FLIP_L interact in cell culture

A and B, interaction of c-FLIP_L and DR5 proteins in co-immunoprecipitation studies. HEK 293 cells were co-transfected with expression vectors for vector alone, FLAG-tagged c-FLIP_L, DR5, or c-FLIP_L and DR5 as indicated. FLAG immunoprecipitates were analyzed for the presence of DR5 and DR5 immunoprecipitates were analyzed for the presence of c-FLIP_L. The presence of DR5 and c-FLIP in cell extracts were verified by immunoblotting for DR5 or c-FLIP. C, interaction between c-FLIP_p12 and DR5 in co-immunoprecipitation studies. HEK 293 cells were co-transfected with expression vectors for vector alone, FLAG-tagged c-FLIP_p12, DR5, or c-FLIP₁₂ and DR5 as indicated. FLAG immunoprecipitates were analyzed for the presence of DR5 and DR5 immunoprecipitates were analyzed for the presence of c-FLIP_p12. The presence of DR5 in total cell lysate is shown by Western blot analysis with an anti-Myc antibody. D, interaction between endogenous c-FLIP_L and endogenous DR5 is independent of FADD. Association of endogenous DR5 with endogenous c-FLIP_L in parental Jurkat A3 and FADD-deficient Jurkat A3 cells is shown by immunoprecipitation with DR5 antibody followed by immunoblotting with c-FLIP antibody. The level of DR5 protein in the immunocomplex is determined by immunoblotting with DR5 antibody. An unrelated immune serum is used as a negative control. In the bottom panel, the level of FADD expression is shown for each cell line.

FIG. 4. c-FLIP_L interacts with DR5 in the absence of TRAIL stimulation and upon formation of the FADD/caspase-8 DISC, DR5, and c-FLIP_L interaction is diminished

Time course of c-FLIP_L and DR5 interaction and FADD/caspase-8 DISC formation was carried out in Jurkat cells that were treated with 10 ng/ml of TRAIL for 0, 5, 15, 30, 60, and 120 min. In stimulated cells, upon TRAIL treatment (10 ng/ml) for 1 h, cells were lysed and the assembled DISCs were immunoprecipitated with protein A and analyzed by Western blotting using antibodies to FADD, caspase-8, caspase-10, TRADD, c-FLIP_L, c-FLIP_p43, and c-FLIP_s In unstimulated condition, cells were first lysed, and then TRAIL was added followed by analysis of the immunocomplex. In the absence of TRAIL treatment, c-FLIP_L is associated with TRAIL receptor. Approximately, 5 min after TRAIL treatment FADD and caspase-8 are recruited to the DISC. At the same time, the level of c-FLIP_L is diminished and the levels of c-FLIP_p43 and c-FLIP_S are increased in the complex.

FIG. 5. A cellular-membrane permeable version of the DR5 binding domain of c-FLIP triggers apoptosis

A–C, Jurkat cells were incubated in medium containing vehicle (Mock), 15 µm TAT, or 15 µm TAT-FLIP cellular membrane permeable peptides for 2 h (A and C) or 16 h (B). A, immunofluorescence analysis demonstrated that TAT and TAT-FLIP are internalized. B, cells were analyzed for the percentage of apoptotic sub-G₁ population by flow cytometry. M₁ is the G₀/G₁ apoptotic population. C, percent apoptosis was also determined by annexin V-PE staining and flow cytometry. Apoptotic rates from three independent experiments are shown in B and C.