Fas-associated protein with death domain (FADD)-independent recruitment of c-FLIPL to death receptor 5.
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.
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215, USA.
Tai-Guang
TG
Jin
Alexei
A
Kurakin
Nordine
N
Benhaga
Karon
K
Abe
Mehrdad
M
Mohseni
Ferry
F
Sandra
Keli
K
Song
Brian K
BK
Kay
Roya
R
Khosravi-Far
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000502:physiology*; D000409:Alanine / Q000737:chemistry; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D019169:Jurkat Cells; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry*; D053221:TNF-Related Apoptosis-Inducing Ligand; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D019151:Peptide Library; D010455:Peptides; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D053221:TNF-Related Apoptosis-Inducing Ligand; C505639:TNFRSF10B protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D005982:Glutathione Transferase; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D000409:Alanine