CFLAR (CASP8 and FADD-like apoptosis regulator)

2008-09-01   Christophe Le Clorennec , Daniel B Longley , Timothy Wilson 

Drug Resistance Group, Centre for Cancer Research, Cell Biology, Queens University Belfast, Belfast, Northern Ireland




Atlas Image
Genomic Organization and splice variants of c-FLAR (c-FLIP) gene. Schematic representation of the structure of the 48kb c-FLAR gene, which contains 14 exons and is transcribed into 11 alternative splice forms. The start and stop sites for translation of the various splice forms are indicated as arrowheads and asterisks, respectively. Only 3 proteins are expressed at the protein level: FLIPS, FLIPR and FLIPL (adapted from Djerbi M et al 2001).


14 exons; DNA Size 48kb.


FLIPL: mRNA Size: 2243 nucleotides (nt); coding sequence: 1443 nt; FLIPS: mRNA size: 1062 nt; coding sequence: 666 nt.



In 1997, a new family of viral genes encoding viral FLIP (v-FLIP, Fas-associated death domain (FADD)-like interleukin-1 beta converting enzyme (FLICE) inhibitory protein) were identified as proteins containing the Death Effector Domain (DED) which interact with certain caspases: caspase 8 (also termed FLICE) and caspase 10 (Hofmann, 1999). These proteins are principally composed of two homologous DED regions, which are found in a wide family of DED-containing proteins, including procaspase-8, procaspase-10 and FADD, which are components of the DISC (Death Inducing Signalling Complex) formed by death receptors such as Fas (CD95), DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (Ashkenazi and Dixit, 1999). The v-FLIP proteins were first identified in gamma-herpesviruses, such as the Kaposi-associated human herpesvirus-8 (HHV-8), the equine herpesvirus-2 (EHV-2), the herpesvirus saimiri (HVS) and found in the rhesus rhadinovirus (RRV) (Bertin et al., 1997; Hu et al., 1997; Searles et al., 1999; Thome et al., 1997). Two additional v-FLIP variants with carboxy-terminal extensions of unknown function are found in the human molluscipoxvirus (MCV) (Bertin et al., 1997; Hu et al., 1997; Thome et al., 1997).
Soon after the discovery of v-FLIP proteins, the mammalian cellular counterparts were identified, and called c-FLIP proteins (also called CASH, Casper, CLARP, FLAME, I-FLICE, MRIT or usurpin). Among 13 distinct c-FLIP splice variants which have been reported, only three are expressed as proteins: the 55 kDa long form (c-FLIPL), the 26 kDa short form (c-FLIPS) and the 24 kDa short form of c-FLIP (c-FLIPR), identified in the Raji B-cell line (Golks et al., 2005; Budd et al., 2006).
Atlas Image
Overview of c-FLIP isoforms and v-FLIP isoforms. All the c-FLIP proteins carry two tandem death effector domains (DEDs). c-FLIPL also contains a caspase 8-like domain. The sites cleaved by procaspase-8 or by active caspase-8 are shown. Total number of amino acids is given.


c-FLIPL is composed of 480 amino acids and contains a longer carboxy-terminus than cFLIPS. c-FLIPL closely resembles the overall structure of procaspase-8 and procaspase-10 (Figure 2). c-FLIPL contains two DEDs followed by a caspase-like domain. However, the C-terminal caspase-like domain of c-FLIPL lacks caspase enzymatic activity, owing to the substitution of several amino acids, including the crucial cysteine residue in the Gln-Ala-Cys-X-Gly motif (X: any amino acid) and the histidine residue in the His-Gly motif (Cohen, 1997). These two residues are necessary for caspase catalytic activity and are conserved in all caspases. c-FLIPL contains two conserved aspartic-acid cleavage sites: Asp-198, between DED2 and the p20-like domain; and Asp-376, between the p20- and p10-like domains, both of which can be cleaved during Fas- and TRAIL-induced apoptosis (Irmler, 1997; Scaffidi et al., 1999; Golks et al., 2006). This leads to the generation of p43-FLIP, which is implicated in the activation of different signalling pathways such as NF-kappa B pathway (Kataoka and Tschopp, 2004). In addition to NF-kappaB signaling, c-FLIPL has also been shown to activate Erk signaling pathway by binding to Raf-1 (Kataoka, 2000; Park et al., 2001).

The short form c-FLIPS is composed of 221 amino acids and has the same structure as vFLIP proteins, except that in addition to the two DEDs of cFLIPS, a carboxy-terminal tail composed of approximatively 20 amino acids is present that seems to be crucial for its ubiquitinylation and subsequent proteasomal degradation (Poukkula et al., 2005).

The short form c-FLIPR is composed of 213 amino acids, contains two DEDs and lacks the additional carboxy terminal amino acids present in c-FLIPS (Golks et al., 2005).

Atlas Image
Schematic diagram of c-FLIP recruitment to the DISC. All the c-FLIP proteins carry two tandem death effector domains (DEDs), which can bind FADD and procaspase-8. c-FLIPL is structurally very similar to procaspase-8 apart from the active site of c-FLIP in which cysteine 360 has been substituted by a tyrosine, and in another active site, histidine 317 has been substituted by an arginine in c-FLIPL.


c-FLIPL is expressed in many tissues, most abundantly in the heart, skeletal muscle, lymphoid tissues and kidney. c-FLIP is abundantly and constitutively expressed in a wide array of normal cell types, including neurons, cardiac myocytes, endothelial cells, keratinocytes, pancreatic beta cells, dendritic cells (DCs), macrophages, CD34+ haematopoietic stem cells and spermatocytes (Ashany et al., 1999; Bouchet, 2002; Davidson et al., 2003; Desbarats, 2003; Giampietri, 2003; Kiener, 1997; Kim et al., 2002; Maedler, 2002; Marconi, 2004; Rescigno, 2000). c-FLIP is highly expressed in various types of tumour cells, including colorectal carcinoma (Ryu et al., 2001; Ullenhag et al., 2007), gastric carcinoma (Nam et al., 2003; Zhou et al., 2004), pancreatic carcinoma (Elnemr et al., 2001), Hodgkins lymphoma (Dutton et al., 2004; Mathas et al., 2004; Thomas et al., 2002), B cell chronic lymphocytic leukemia (MacFarlane et al., 2002; Olsson et al., 2001), melanoma (Griffith et al., 1998), ovarian carcinoma (Abedini et al., 2004; Mezzanzanica et al., 2004), cervical carcinoma (Wang et al., 2007), bladder urothelial carcinoma and prostate carcinoma (Korkolopoulou et al., 2004; Zhang et al., 2004). All of these tumours are often resistant to death receptor-mediated apoptosis. The expression of c-FLIP has been proven to be one of the major determinants of the resistance to death ligands such as FasL and TRAIL (TNF-related apoptosis-inducing ligand), and numerous reports have shown that down-regulation of c-FLIP results in sensitizing various resistant tumour cells to death ligands (Kim et al., 2000; Longley et al., 2006; Ricci et al., 2004; Wilson et al., 2007).


c-FLIP proteins are localized in the cytosol.


In many studies, in vitro, FLIP proteins (v-FLIP, c-FLIPR, c-FLIPS and c-FLIPL) protect cells against apoptosis induced by several death receptors, including FAS, tumour-necrosis factor (TNF) receptor 1 ( TNFR1 ), TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (TRAILR1; also known as DR4), TRAILR2 (also known as DR5) and TNFR-related apoptosis-mediating protein ( TRAMP; also known as DR3). Due to its high structural homology with procaspase-8, FLIP interferes with caspase-8 activation at the death-inducing signalling complex (DISC), which is formed after death receptor ligation (Ashkenazi and Dixit, 1999). The inhibition of Death Receptor-mediated apoptosis by FLIP is due to competition between the DEDs of FLIP and procaspase-8/10 for recruitment to the adaptor protein FADD at the DISC (Irmler, 1997; Srinivasula, 1997). Procaspase-8 recruitment to the DISC results in its homodimerization and two sequential cleavage steps that generate p10 and p18 fragments that heterodimerize to form fully active (p10-p18)2 caspase-8 that dissociates from the DISC (Krammer et al., 2007).

When the death receptors are stimulated by their corresponding ligand, they recruit the adapter molecule FADD. FADD can then recruit DED containing proteins, e.g. caspase-8, and form a DISC. c-FLIP inhibits caspase-8 activation at the DISC. c-FLIPL and c-FLIPS have been shown to block death receptor-mediated apoptosis by forming a proteolytically inactive heterodimer with procaspase-8 (Golks et al., 2005; Krueger et al., 2001). However, cleavage is blocked at different stages. For c-FLIPS and c-FLIPR, both cleavage steps required for procaspase-8 activation are completely blocked. In contrast, c-FLIPL allows partial cleavage of procaspase-8 at the DISC (Figure 3). When a molecule of procaspase-8 and c-FLIPL come into contact at the DISC, a conformational change in the two molecules occurs. This leads to the autocatalytic cleavage of the p10 subunit from procaspase-8. c-FLIPL is also partially cleaved by the procaspase-8 molecule to generate a p12 subunit. However, cleavage is stopped at this stage and no p18 subunit is generated from caspase-8. It has been hypothesised that the second reciprocal trans-catalytic cleavage step cannot occur because of the lack of the cysteine residue at the active site of c-FLIPL (Micheau, 2002). The resulting cleavage products are p41/43- and p10-caspase-8 products; and p43- and p12-c-FLIPL intermediates. Furthermore, Kreuger et al demonstrated that the p41/43-caspase-8 and p43-c-FLIPL intermediates remain bound at the DISC (Krueger et al., 2001). Recently, it has been proposed that the DISC-bound caspase 8/FLIP complex has catalytic activity that is not capable of generating a pro-apoptotic signal, but that can cleave local substrates such as RIP (receptor-interacting protein) (Micheau, 2002).

Implicated in

Entity name
Classical Hodgkins lymphoma (cHL), a common human lymphoma, has been proposed to be derived from germinal centre (GC) B cells in the majority of cases (Kuppers et al., 2002). Among tumour-forming cells, the malignant Hodgkin/Reed-Sternberg (HRS) cells, which represents the malignant population of cHD disease, are rare and represent only 1% of cells in affected lymph nodes. HRS cells have lost their B cell phenotype, including immunoglobulin (Ig) expression (Schwering et al., 2003). Usually, B cells with non-functional Ig expression undergo apoptosis.
Hodgkin/Reed-Sternberg (HRS) cells are most often resistant to Death receptor-mediated apoptosis such as is mediated by FasL or TRAIL. The expression of c-FLIP has been proven to be one of the major determinants of this resistance. HRS cells have been shown to overexpress c-FLIP proteins in a NF-kappa B-dependent manner. Some studies have shown that the high level of c-FLIP prevent the activation of caspase-8 by inhibition of procaspase-8 processing. To remove this resistance to Death receptor mediated apoptosis, some reports have shown that specific down-regulation of c-FLIP by small interfering RNA oligoribonucleotides strategies is sufficient to sensitize HRS cells to Fas and TRAIL-induced apoptosis (Mathas et al., 2004).
Entity name
Colorectal cancer (CRC)
Colorectal cancer is a major cause of cancer mortality. Response rates in the advanced disease setting are of the order of 45% to 50% for the most effective drug combinations. Drug resistance is a major problem in this disease (and other cancers) and is often the result of insufficient apoptosis induced by chemotherapy.
Clinical studies have demonstrated significantly elevated c-FLIP expression in colorectal tumours (Ryu et al., 2001), suggesting that c-FLIP may be play a role in the pathogenesis of this disease. Indeed, c-FLIP(L) overexpression was associated with poor prognosis in colorectal cancer patients (Ullenhag et al., 2007).
Entity name
Graves disease
Graves disease is an autoimmune form of hyperthyroidism. In the context of this disease, lymphocyte TH2 cells infiltrate the thyroid gland and, via production of IL4 and IL10, stimulate thyrocytes to become more resistant to Fas-mediated apoptosis, in part by upregulation of c-FLIP and Bcl-XL (Stassi, 2000).
Entity name
Multiple sclerosis (MS)
Multiple sclerosis (MS): a neuroinflammatory disease that is thought to have an autoimmune basis due to autoreactive T cells responding to myelin self-antigens (Conlon et al., 1999). Autoimmune diseases such as MS can result from the lack of elimination of pathogenic, autoreactive lymphocytes.
In this disease, the pathological upregulation of FLIP levels in T cells might contribute to the accumulation of lymphocytes in cortical-spinal-fluid and accumulation of activated-peripheral T cells in patients with clinically active MS. FLIPL and FLIPS were found to be specifically overexpressed in T cells of MS patients, indicating that abnormally high FLIP expression levels might extend the viability of potentially pathogenic, autoreactive T cells in the context of this disease (Semra et al., 2001; Sharief, 2000).


Pubmed IDLast YearTitleAuthors
152585642004Possible role of FLICE-like inhibitory protein (FLIP) in chemoresistant ovarian cancer cells in vitro.Abedini MR et al
102091531999Apoptosis control by death and decoy receptors.Ashkenazi A et al
90370251997Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis.Bertin J et al
124779722002Differential sensitivity of endothelial cells of various species to apoptosis induced by gene transfer of Fas ligand: role of FLIP levels.Bouchet D et al
164984502006cFLIP regulation of lymphocyte activation and development.Budd RC et al
93378441997Caspases: the executioners of apoptosis.Cohen GM et al
104088051999The immunobiology of multiple sclerosis: an autoimmune disease of the central nervous system.Conlon P et al
145967922003FLIP protects cardiomyocytes from apoptosis induced by simulated ischemia/reoxygenation, as demonstrated by short hairpin-induced (shRNA) silencing of FLIP mRNA.Davidson SM et al
125451712003Fas engagement induces neurite growth through ERK activation and p35 upregulation.Desbarats J et al
114391652001Characterization of the human FLICE-inhibitory protein locus and comparison of the anti-apoptotic activity of four different flip isoforms.Djerbi M et al
150965872004Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.Dutton A et al
111725972001Human pancreatic cancer cells disable function of Fas receptors at several levels in Fas signal transduction pathway.Elnemr A et al
127006452003FLIP is expressed in mouse testis and protects germ cells from apoptosis.Giampietri C et al
157016492005c-FLIPR, a new regulator of death receptor-induced apoptosis.Golks A et al
97433431998Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells.Griffith TS et al
104420921999The modular nature of apoptotic signaling proteins.Hofmann K et al
90924881997A novel family of viral death effector domain-containing molecules that inhibit both CD-95- and tumor necrosis factor receptor-1-induced apoptosis.Hu S et al
92171611997Inhibition of death receptor signals by cellular FLIP.Irmler M et al
108372472000The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways.Kataoka T et al
150240542004N-terminal fragment of c-FLIP(L) processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF-kappaB signaling pathway.Kataoka T et al
91269331997Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages.Kiener PA et al
118978742002Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis.Kim H et al
106905082000Molecular determinants of response to TRAIL in killing of normal and cancer cells.Kim K et al
151839892004c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations.Korkolopoulou P et al
175895432007Life and death in peripheral T cells.Krammer PH et al
112792182001Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex.Krueger A et al
120788902002Biology of Hodgkin's lymphoma.Küppers R et al
162474742006c-FLIP inhibits chemotherapy-induced colorectal cancer cell death.Longley DB et al
123604072002Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia.MacFarlane M et al
120607682002FLIP switches Fas-mediated glucose signaling in human pancreatic beta cells from apoptosis to cell replication.Maedler K et al
155074842004FLICE/caspase-8 activation triggers anoikis induced by beta1-integrin blockade in human keratinocytes.Marconi A et al
150788992004c-FLIP mediates resistance of Hodgkin/Reed-Sternberg cells to death receptor-induced apoptosis.Mathas S et al
152974242004CD95-mediated apoptosis is impaired at receptor level by cellular FLICE-inhibitory protein (long form) in wild-type p53 human ovarian carcinoma.Mezzanzanica D et al
122154472002The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.Micheau O et al
146620222003Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosis in human gastric cancers.Nam SY et al
117536072001Sensitization to TRAIL-induced apoptosis and modulation of FLICE-inhibitory protein in B chronic lymphocytic leukemia by actinomycin D.Olsson A et al
117413212001Alternative splicing variants of c-FLIP transduce the differential signal through the Raf or TRAF2 in TNF-induced cell proliferation.Park SJ et al
158862052005Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.Poukkula M et al
111048082000Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses.Rescigno M et al
153676742004Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity.Ricci MS et al
175136032007Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells.Rogers KM et al
113291362001Increased expression of cFLIP(L) in colonic adenocarcinoma.Ryu BK et al
98805311999The role of c-FLIP in modulation of CD95-induced apoptosis.Scaffidi C et al
123937312003Loss of the B-lineage-specific gene expression program in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.Schwering I et al
100741541999Sequence and genomic analysis of a Rhesus macaque rhadinovirus with similarity to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8.Searles RP et al
111649112001Overexpression of the apoptosis inhibitor FLIP in T cells correlates with disease activity in multiple sclerosis.Semra YK et al
110638392000Increased cellular expression of the caspase inhibitor FLIP in intrathecal lymphocytes from patients with multiple sclerosis.Sharief MK et al
92280181997FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis.Srinivasula SM et al
111018692000Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins.Stassi G et al
119437362002Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells.Thomas RK et al
90874141997Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.Thome M et al
177855592007Overexpression of FLIPL is an independent marker of poor prognosis in colorectal cancer patients.Ullenhag GJ et al
174338272007The relationship between c-FLIP expression and human papillomavirus E2 gene disruption in cervical carcinogenesis.Wang W et al
175751422007c-FLIP: a key regulator of colorectal cancer cell death.Wilson TR et al
154662042004Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer.Zhang X et al
146361562004Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma.Zhou XD et al

Other Information

Locus ID:

NCBI: 8837
MIM: 603599
HGNC: 1876
Ensembl: ENSG00000003402


dbSNP: 8837
ClinVar: 8837
TCGA: ENSG00000003402


Gene IDTranscript IDUniprot

Expression (GTEx)



PathwaySourceExternal ID
Autophagy - animalKEGGko04140
Autophagy - animalKEGGhsa04140
Chagas disease (American trypanosomiasis)KEGGko05142
Chagas disease (American trypanosomiasis)KEGGhsa05142
NF-kappa B signaling pathwayKEGGhsa04064
NF-kappa B signaling pathwayKEGGko04064
TNF signaling pathwayKEGGhsa04668
TNF signaling pathwayKEGGko04668
Signal TransductionREACTOMER-HSA-162582
Death Receptor SignallingREACTOMER-HSA-73887
TRAIL signalingREACTOMER-HSA-75158
Programmed Cell DeathREACTOMER-HSA-5357801
Caspase activation via extrinsic apoptotic signalling pathwayREACTOMER-HSA-5357769
Ligand-dependent caspase activationREACTOMER-HSA-140534
Dimerization of procaspase-8REACTOMER-HSA-69416
Regulation by c-FLIPREACTOMER-HSA-3371378
Regulated NecrosisREACTOMER-HSA-5218859
RIPK1-mediated regulated necrosisREACTOMER-HSA-5213460
Regulation of necroptotic cell deathREACTOMER-HSA-5675482
CASP8 activity is inhibitedREACTOMER-HSA-5218900

Protein levels (Protein atlas)

Not detected


Pubmed IDYearTitleCitations
118305872002The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex.103
150240542004N-terminal fragment of c-FLIP(L) processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF-kappaB signaling pathway.85
199131212009Gene-centric association signals for lipids and apolipoproteins identified via the HumanCVD BeadChip.85
157016492005c-FLIPR, a new regulator of death receptor-induced apoptosis.84
119406022002An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL-induced apoptosis.74
174501412007A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers.74
226832652012Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.70
162474742006c-FLIP inhibits chemotherapy-induced colorectal cancer cell death.68
269909872016Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate.65
158862052005Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.60


Christophe Le Clorennec ; Daniel B Longley ; Timothy Wilson

CFLAR (CASP8 and FADD-like apoptosis regulator)

Atlas Genet Cytogenet Oncol Haematol. 2008-09-01

Online version: