Cellular FLIP Inhibits β-Catenin Ubiquitylation and Enhances Wnt Signaling

Abstract

Ligation of death receptors, such as Fas, triggers apoptosis in many types of cells (28), which is inhibited by cellular FLIP (cFLIP, also known as I-FLICE, FLAME-I, Casper, CASH, MRIT and Usurpin) (8,11,13,16,34,40,41,44). The long form of cFLIP (cFLIP-L) is highly homologous to caspase 8, containing two death effector domains (DED) and a caspaselike domain at the amino and carboxy termini, respectively. cFLIP-L, however, does not have caspase activity due to the lack of a conserved cysteine residue in the caspase-like domain. Upon death receptor ligation, cFLIP-L is recruited to the death receptor complex, together with FADD and caspase 8, and inhibits apoptosis signaling. cFLIP-L is expressed in various cancers (2,16,27,36,43,44), which suggests a role for cFLIP-L in protecting cancer cells from cellular immunity using the Fas system (5, 6, 25).cFLIP, however, does not always inhibit apoptosis signaling but also mediates growth signals in some cases. Under conditions in which the proliferation of CD3-activated human T lymphocytes is increased by recombinant Fas ligand, cFLIP-L interacts with tumor necrosis factor receptor-associated factors 1 and 2, as well as the kinases RIP and Raf-1, resulting in the activation of the NF-B and ERK signaling pathways (17). The ability of cFLIP-L to switch Fas-mediated glucose signaling from a death signal into a proliferation signal in human pancreatic ␤ cells was also reported (24).The Wnt signal transduction pathway plays a critical and evolutionarily conserved role during embryogenesis (3, 10, 46). The Wnt signal is mediated by ␤-catenin, a transcription factor that is normally degraded by the ubiquitin-proteasome system in cytosol. Phosphorylation of ␤-catenin by a large protein complex involving adenomatous polyposis coli (APC) protein, Axin, and glycogen synthase kinase 3␤ (GSK3␤) initiates the ubiquitylation and proteasomal degradation of ␤-catenin (15, 49). Upon Wnt signaling, phosphorylation of ␤-catenin is inhibited, which results in the accumulation and translocation of ␤-catenin into nuclei, thereby inducing the expression of several genes, such as c-myc and the cyclin D gene. Mutations in APC, Axin, and ␤-catenin genes resulting in abolished ␤-catenin ubiquitylation are found in many human cancers (4,9,20,26,30,32,35,37), indicating that inappropriate activation of Wnt signaling plays an important role in human cancers (31,33).In this paper, we report that cFLIP-L inhibits ␤-catenin ubiquitylation and enhances Wnt signaling, which suggests an additional mechanism involved in tumorgenesis, in addition to inhibiting apoptosis signaling. MATERIALS AND METHODSPlasmids. Human cFLIP-L and a short splicing variant of cFLIP (cFLIP-S) were amplified by PCR from a Jurkat cDNA library and subcloned into pcDNAbased mammalian expression vectors (Invitrogen). For deletion mutant constructs, DNA sequences corresponding to different regions of cFLIP-L were

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis

“…We were not able to detect p22 of cFLIP L and cFLIP S in the TRAIL or CD95L DISC of cFLIP L -or cFLIP S -expressing melanoma cells, suggesting cell-type specific differences when compared to the published data (Golks et al, 2006). In addition, it is tempting to speculate that such cFLIP fragments or uncleaved cFLIP L itself may modulate important apoptosis-independent signaling pathways such as Wnt (Naito et al, 2004) cytotoxic agents (Ivanov and Hei, 2006) or imatinib (Hamai et al, 2006) sensitized resistant melanomas to TRAIL-mediated apoptosis. However, transient transfection studies might be hampered by the activation of cellular responses due to toxic effects of the transfection procedure.…”

Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L

“…We, and others, previously reported that cFLIP-L enhances Wnt signaling by inhibiting ubiquitylation of -catenin, which is a mediator of canonical Wnt signaling (Naito et al, 2004;Nakagiri et al, 2005). In unstimulated cells, free cytosolic -catenin is maintained at a low level by serine or threonine phosphorylation of -catenin, followed by ubiquitylation and degradation by the proteasome.…”

FLIP and the death effector domain family

“…At another level, undoubtedly more proteins will be added to the mix that comprise DED-mediated signaling platforms (such as TRAF1, TRAF2 and Raf1 as mentioned above and others), and elucidating if and how these proteins integrate with any of the core DED family members to activate various signaling pathways will represent another initial yet crucial step in answering the posed questions. Along with these challenges, new and exciting frontiers involving DED family members are being discovered and a few examples include: induction of programmed cell death from locations outside of death receptors including unligated integrins (Stupack et al, 2001(Stupack et al, , 2006 and the mitochondria (Lee et al, 2007), regulation of autophagy (and autophagic cell death) by caspase-8 and FADD (Yu et al, 2004;Pyo et al, 2005;Thorburn et al, 2005), regulation of JNK (Nakajima et al, 2006) and WNT signaling by c-FLIP L (Naito et al, 2004;Nakagiri et al, 2005), and modulation of gene expression from the androgen receptor (Qi et al, 2007) and p53 by procaspase-8 (Yao et al, 2007). The increasingly complex involvement of DED proteins in different pathways and cellular processes is indeed unfolding from ongoing effort, and additional answers that verify and map out these new connections lie ahead.…”