Research Article 23 Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L

Ryohei Katayama1,2, Toshiyasu Ishioka1, Shinji Takada3, Ritsuko Takada3, Naoya Fujita2, Takashi Tsuruo2 and Mikihiko Naito1,4,* 1Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan 2Cancer Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan 3Okazaki Institute for Integrative Biosciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan 4National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan *Author for correspondence ([email protected])

Accepted 2 October 2009 Journal of Cell Science 123, 23-28 Published by The Company of Biologists 2010 doi:10.1242/jcs.058602

Summary Cellular FLIP (cFLIP) inhibits the signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) enhances Wnt signaling via inhibition of -catenin ubiquitylation. In this report, we present evidence that cFLIP-L translocates into the nucleus, which could have a role in modulation of Wnt signaling. cFLIP-L has a functional bipartite nuclear localization signal (NLS) at the C-terminus. Wild-type cFLIP-L (wt-FLIP-L) localizes in both the nucleus and cytoplasm, whereas NLS-mutated cFLIP-L localizes predominantly in the cytoplasm. cFLIP-L also has a (NES) near the NLS, and B, an inhibitor of CRM1-dependent nuclear export, increases the nuclear accumulation of cFLIP-L, suggesting that it shuttles between the nucleus and cytoplasm. Expression of mutant cFLIP-L with a deletion or mutations in the NLS and NES confers resistance to Fas-mediated apoptosis, as does wt-FLIP-L, but they do not enhance Wnt signaling, which suggests an important role of the C-terminus of cFLIP-L in Wnt-signaling modulation. When wt-FLIP-L is expressed in the cytoplasm by conjugation with exogenous NES (NES-FLIP-L), Wnt signaling is not enhanced, whereas the NES-FLIP-L increases cytoplasmic -catenin as efficiently as wt-FLIP-L. cFLIP-L physically interacts with the reporter plasmid for Wnt signaling, but not with the control plasmid. These results suggest a role for nuclear cFLIP-L in the modulation of Wnt signaling.

Key words: FLIP, NLS, Wnt signaling

Introduction revealed the presence of a short isoform of FLIP , cFLIP-S,

Journal of Cell Science Nucleocytoplasmic shuttling of proteins has an important role in and both isoforms of cFLIP can inhibit death-receptor-mediated cell function (Weis, 1998). The coordination of trafficking between apoptosis. In addition to apoptosis inhibition, cFLIP-L mediates the the nucleus and cytoplasm is determined by the balance of nuclear activation of NF-B, PI3K and Erk by virtue of its capacity to recruit import and export activity (Hood and Silver, 1999; Mattaj and the adaptor proteins involved in each signaling pathway, such as Englmeier, 1998; Nigg, 1997). Proteins destined for transport into TRAF-1, TRAF-2, RIP and Raf-1 (Chaudhary et al., 2000; Fang et the nucleus contain amino acid targeting sequences called nuclear al., 2004; Kataoka et al., 2000; Kataoka and Tschopp, 2004). localization signals (NLSs), which principally consist of either one Inhibition of the JNK pathway by direct binding to MKK7 has also (monopartite) or two (bipartite) stretches of basic amino acids been reported (Nakajima et al., 2006). All of these phenomena take (Lange et al., 2007). By contrast, proteins that are exported from place in the cytoplasm, and therefore, cFLIP is regarded as a the nucleus mainly contain nuclear export signal (NES) sequences, cytoplasmic protein. which consist of several residues distributed with an uneven We, and others, previously reported that cFLIP-L enhances Wnt spacing (Fischer et al., 1995; Wen et al., 1995). A related family signaling by inhibiting ubiquitylation of -catenin, which is a of shuttling transport factors, the importins and exportins, recognize mediator of canonical Wnt signaling (Naito et al., 2004; Nakagiri the NLS- or NES-containing proteins and mediate nuclear import et al., 2005). In unstimulated cells, free cytosolic -catenin is or export, respectively. Leptomycin B (LMB) effectively blocks maintained at a low level by serine or threonine protein export from the nucleus by directly binding to exportin-1 of -catenin, followed by ubiquitylation and degradation by the (CRM1) (Fukuda et al., 1997). proteasome. When cells are stimulated with Wnt3a, the Cellular FLIP (cFLIP, also known as I-FLICE, FLAME1, Casper, phosphorylation of -catenin is inhibited, resulting in the CASH, MRIT and Usurpin) is an inhibitor of the apoptosis initiated accumulation and translocation of -catenin into the nucleus. In the by death receptor ligation (Irmler et al., 1997). The long form of nucleus, -catenin interacts with T-cell-factor/lymphoid-enhancing c-FLIP (cFLIP-L) is highly homologous to caspase-8, and contains factor (TCF/LEF), and activates their target genes to promote cell two death effector domains (DED) and a caspase-like domain at growth (Clevers, 2006; Logan and Nusse, 2004). Exogenously the N- and C-termini, respectively. cFLIP-L, however, does not have expressed and endogenous cFLIP-L expressed in some cancer cells caspase activity because it has no conserved residue in the are prone to aggregate in the cells and inhibit the ubiquitylation of caspase-like domain. Upon death receptor ligation, cFLIP-L is short-lived proteins, including -catenin, resulting in an recruited to the death receptor complex, together with FADD and enhancement of Wnt signaling (Ishioka et al., 2007; Naito et al., caspase-8, and inhibits apoptosis signaling. Previous studies 2004). 24 Journal of Cell Science 123 (1)

In this study, we show that cFLIP-L has NLS and NES sequences indicate that cFLIP-L localizes in both the nucleus and cytoplasm, at the C-terminus, and shuttles between the nucleus and the whereas cFLIP-S is exclusively found in the cytoplasm. cytoplasm. Forced expression of cFLIP-L in the cytoplasm by conjugation of NES to cFLIP-L abrogated the ability of cFLIP-L Leptomycin B increases nuclear localization of cFLIP-L to enhance Wnt signaling, while retaining the capacity to increase It is known that many proteins localizing in both the nucleus and the -catenin protein level. In addition, cFLIP-L physically cytoplasm shuttle between the two compartments. The transport interacted with the reporter plasmid for Wnt signaling. These results from the nucleus to the cytoplasm is often mediated by CRM1 suggest that nuclear cFLIP-L has a role in modulating Wnt signaling. (Fornerod et al., 1997; Gama-Carvalho and Carmo-Fonseca, 2001; Harel and Forbes, 2004; Stade et al., 1997), which can be inhibited Results specifically by an , leptomycin B (LMB) (Fasken et al., Nuclear and cytoplasmic localization of cFLIP-L 2000; Kudo et al., 1999; Kudo et al., 1998; Wolff et al., 1997). To After ligation of death receptor, cFLIP-L is recruited to the death- examine whether cFLIP-L is exported in a CRM1-dependent inducing signaling complex (DISC) composed of death receptor, manner, we generated HT1080 cells constitutively expressing FADD and caspase-8, and therefore, cFLIP-L is regarded as a EGFP-cFLIP-L, and examined the localization of the EGFP-cFLIP- cytoplasmic protein. However, when cells constitutively expressing L with or without LMB. The EGFP-cFLIP-L exhibited cytoplasmic Myc-tagged cFLIP-L were stained with anti-Myc , a strong and nuclear localization without LMB treatment, and the signal was observed in nuclei (Fig. 1A). Biochemical fractionation accumulation of the protein in nuclei was increased after treatment also showed that a significant amount of Myc-tagged cFLIP-L was with LMB (Fig. 2A). Nuclear and cytoplasmic fractionation of the extracted in the nuclear fraction (Fig. 1B). We also examined the cells confirmed the nuclear accumulation of cFLIP-L (~1.6-fold localization of endogenous cFLIP protein by immunocytochemical increase) by LMB treatment (Fig. 2B). On the other hand, EGFP- staining in A549 cells, and found cFLIP protein distributed in the cFLIP-S protein localizes abundantly in the cytoplasm, and the cytoplasm and nuclei (Fig. 1C). Cytoplasmic and nuclear pattern of distribution did not change after LMB treatment (Fig. fractionation of cancer cells expressing the cFLIP protein (A549, ACHN and DU145) showed that a significant amount of cFLIP-L was extracted in the nuclear fraction, whereas cFLIP-S was predominantly in the cytoplasmic fraction (Fig. 1D). These results Journal of Cell Science

Fig. 2. Leptomycin B increases nuclear localization of cFLIP-L. Fig. 1. Nuclear and cytoplasmic localization of cellular FLIP-L. (A)HT1080 cells constitutively expressing EGFP-cFLIP-L were incubated (A)HT1080 cells constitutively expressing wild-type cFLIP-L were fixed with with or without 10 ng/ml Leptomycin B (LMB) for 4 hours. The cells were 4% formaldehyde and immunostained with an anti-FLIP antibody (NF-6), observed under confocal microscopy. Scale bar: 20m. (B)HT1080 cells followed by staining with Alexa-Fluor-488-conjugated anti-mouse IgG constitutively expressing EGFP-cFLIP-L or EGFP-cFLIP-S were incubated secondary antibody. (B)The cFLIP-L stable transfectants were fractionated with or without LMB. Cells were fractionated into cytosol and nucleus into cytosol and nucleus fractions, and analyzed by immunoblotting with the fractions, and analyzed by western blotting with the indicated . indicated antibodies. (C)A549 cells were immunostained with anti-FLIP (C)A549 cells treated with LMB for 6 hours were fractionated into cytosol antibody. Scale bars: 20m. (D)A549 lung cancer, ACHN renal carcinoma and nucleus fractions, and analyzed by western blotting with the indicated and DU145 prostate cancer cells were fractionated into cytosol and nucleus antibodies. The numbers under the upper panels represent relative intensity of fractions, and 20 from each fraction was analyzed by western the cFLIP band in each fraction (average of two experiments) compared with blotting with the indicated antibodies. C, cytosolic fraction; N, nuclear the corresponding cytosolic fraction (set to 100). C, cytosolic fraction; N, fraction. nuclear fraction. Nuclear cFLIP-L modulates Wnt 25

2B). We also examined the localization of endogenous cFLIP-L in localization. The NLS mutant cFLIP-L proteins predominantly A549 cells treated with LMB. As shown in Fig. 2C, LMB treatment localized in the cytoplasm, whereas the NES mutant localized in increased the nuclear accumulation of endogenous cFLIP-L. the nucleus (Fig. 3B). These results indicate that the localization Collectively, these results suggest that cFLIP-L, but not cFLIP-S, of cFLIP-L is regulated by the NLS and NES sequences at the C- shuttles between the cytoplasm and the nucleus. terminus of cFLIP-L.

Identification of the NLS and NES sequences in cFLIP-L Mutations in the C-terminus of cFLIP-L abrogate the Because cFLIP-L, but not cFLIP-S or a cFLIP-L mutant lacking activity to modulate Wnt signaling 42 amino acids of the C-terminal [cFLIP(1-438)], localized in the As the subcellular localization of a protein is closely related to its nucleus (Figs 1, 2 and supplementary material Fig. S1), we reasoned function (Andersson et al., 2004; Chung and Eng, 2005; Gama- that NLS might be present at the C-terminus of cFLIP-L. We Carvalho and Carmo-Fonseca, 2001; Gomez-Angelats and searched for the NLS-like sequence at the C-terminal region of Cidlowski, 2003; Schickling et al., 2001; Wang et al., 2006; Zhan cFLIP-L, and found a pair of three consecutive basic amino acids et al., 2002), we hypothesized that the NLS and NES mutants of residues (435RKR and 472RKK) separated by 34 amino acids that cFLIP-L might have a different function from wild-type cFLIP-L. could potentially function as an NLS (Fig. 3A, red). We also found Cells constitutively expressing the wild-type and mutant cFLIP-L an NES-like sequence near the putative NLS in cFLIP-L (Fig. 3A, proteins (supplementary material Fig. S2A) displayed resistance to green). Then we examined whether the NLS- and NES-like the apoptosis induced by an anti-Fas antibody (supplementary sequences regulate the nuclear and cytoplasmic localization of material Fig. S2B), but not by an anti-tumor drug, etoposide cFLIP-L by mutation analysis. We generated cells constitutively (supplementary material Fig. S2C), suggesting that anti-apoptotic expressing NLS-mutants (435mt and 472mt) and an NES-mutant function of the NLS- and NES-mutant cFLIP-L proteins is (439mt) of cFLIP-L (Fig. 3A), and examined the protein indistinguishable from that of the wild-type cFLIP-L. We next treated the cells with Wnt3a and measured -catenin-mediated gene expression in the cFLIP-L transfectants. In parental HT1080 cells, Wnt signaling, as measured by the expression of a luciferase reporter gene, was slightly stimulated by treating the cells with Wnt3a in a dose-dependent fashion. This Wnt signaling was greatly enhanced in the wild-type cFLIP-L transfectants, but only marginally in the transfectants expressing NLS and NES mutant cFLIP-L and cFLIP- S proteins (supplementary material Fig. S2D). Similarly, no enhanced Wnt signaling was observed in cells expressing C- terminally deleted cFLIP(1-438) (supplementary material Fig. S3). Transient transfection of wild-type cFLIP-L increased the cellular accumulation of -catenin, and induced -catenin-mediated gene expression, whereas these activities were completely abolished in

Journal of Cell Science the NLS and NES mutant cFLIP-L (supplementary material Fig. S4A,B) and cFLIP(1-438) (supplementary material Fig. S4C,D). These results suggest that the C-terminus of cFLIP-L, which regulates nuclear and cytoplasmic localization by the presence of NLS and NES sequences, is required for the accumulation of endogenous -catenin and the modulation of Wnt signaling.

Role of nuclear cFLIP-L in Wnt signaling To study the role of nuclear cFLIP-L, we conjugated a nuclear export signal sequence of MAPKK to cFLIP-L (NES-FLIP-L, Fig. 4A). The NES-cFLIP-L predominantly localized in the cytoplasm (Fig. 4B), indicating that the NES sequence predominantly determined the cellular distribution of cFLIP-L, even in the presence of the intrinsic NLS at the C-terminus of cFLIP-L. Cells constitutively expressing wild-type and NES-cFLIP-L (Fig. 5A) were comparably resistant to apoptosis induced by Fas antibody (Fig. 5B), and were Fig. 3. cFLIP-L contains a bipartite NLS and an NES at the C-terminus. sensitive to the anti-tumor drug, etoposide (Fig. 5C). We observed (A)Diagram of human cFLIP-L with the positions and amino acid sequences no differences in NF-B activation in cells expressing wild-type, of the two predicted nuclear localization signals (NLSs) and one predicted NLS mutant or NES-cFLIP-L (supplementary material Fig. S5). nuclear export signal (NES). The functionally important basic amino acids of However, Wnt3a-induced gene expression was enhanced in the NLS are shown in red, and the leucine residues of NES are shown in green. wild-type cFLIP-L transfectants, but not in the NES-cFLIP-L Amino acid substitutions to generate NLS and NES mutants are indicated in transfectants (Fig. 5D), although nuclear translocation of -catenin blue and pink, respectively. (B)NLS mutants mainly localize in the cytosol by Wnt3a treatment was equally observed in these cells and the NES mutant mainly localizes in the nucleus. HT1080 cells constitutively expressing EGFP-tagged wild-type and mutant cFLIP-L were (supplementary material Fig. S6). Transient transfection of wild- fixed and immunostained with an anti-FLIP antibody (NF-6), followed by type cFLIP-L and NES-cFLIP-L equally increased -catenin staining with Alexa-Fluor-568-conjugated anti-mouse IgG secondary antibody. accumulation (Fig. 6A), but the -catenin-mediated gene expression The cells were observed under confocal microscopy. Scale bar: 20m. was greatly reduced in the NES-cFLIP-L-expressing cells compared 26 Journal of Cell Science 123 (1)

Fig. 4. NES-cFLIP-L mainly localizes in cytosol. (A)Diagram of NES- cFLIP-L. The NES signal sequence from MAPKK was inserted into the N- terminal of cFLIP-L. (B)Cytoplasmic localization of NES-cFLIP-L. HT1080 cells constitutively expressing wild-type cFLIP-L or NES-cFLIP-L were fixed and stained with an anti-FLIP antibody, and observed under confocal microscopy. Scale bar: 20m.

with the cells expressing wild-type cFLIP-L (Fig. 6B). Although we could not rule out the possibility that the NES sequence conjugated to cFLIP-L interferes with a cytoplasmic function of Fig. 5. NES-cFLIP-L inhibits apoptosis but does not enhance Wnt cFLIP-L that is essential for FLIP-dependent Wnt pathway signaling. (A)Expression of cFLIP-L and NES-cFLIP-L in stable transfectant activation, these results suggest that cFLIP-L activates the Wnt clones. Cell lysates prepared from HT1080 clones constitutively expressing wt-cFLIP-L and NES-cFLIP-L were analyzed by western blotting with signaling pathway through a mechanism independently of -catenin indicated antibodies. ‘Parent’ represents parental HT1080 cells (non- stabilization or downstream of the stabilization, which could take transfected cells). (B,C)NES-cFLIP-L inhibits apoptosis induced by anti-Fas place in the nucleus. antibody. Cells were treated with the indicated concentrations of anti-Fas To study further the role of the nuclear cFLIP-L in Wnt signaling antibody in the presence of 1g/ml cycloheximide (B) or etoposide (C). After modulation, we examined whether cFLIP-L physically interacts with 3 days, viable cells were measured by MTS assay and are expressed as the reporter plasmid for -catenin-mediated gene expression. Fig. percentages compared with untreated cells. (D)NES-cFLIP-L does not

Journal of Cell Science 7 shows that immunoprecipitates of cFLIP-L contained the reporter enhance Wnt signaling. Cells were transfected with a TOP luciferase reporter plasmid TOP-TK-Luc, but not the control plasmid FOP-TK-Luc. plasmid for 12 hours, treated with Wnt3a-conditioned medium for 24 hours, cFLIP-L(1-438), which does not enhance Wnt signaling, was and then cell lysates were analyzed by luciferase assay. Error bars indicate s.d. devoid of this activity. These results suggest a direct role for nuclear cFLIP-L in the modulation of -catenin-mediated gene expression. with DED, because cFLIP-S, which also contains DED and is Discussion recruited to DISC, did not localize in the nucleus (Fig. 1D). cFLIP- Nucleocytoplasmic transportation of proteins has an important role L also interacts with other DED-containing proteins, such as in the regulation of many cellular processes. Previous studies DEDD1 and DEDD2, which are nuclear proteins that sequester reported that cFLIP-L is a cytoplasmic protein that inhibits the cFLIP in the nucleus upon overexpression in human cell lines (Zhan apoptosis signaling initiated by death receptor ligation at the cell et al., 2002). However, DEDD2 overexpression sequestered the membrane. In this study, we report for the first time that cFLIP-L cFLIP protein in nucleoli, whereas the wild-type and NES-mutant localizes in the nucleus and cytoplasm, whereas cFLIP-S cFLIP-L proteins localized in the nucleoplasm (Figs 1 and 2), predominantly localizes in the cytoplasm. We identified NLS and suggesting that DEDD2 is not involved in the nuclear localization NES sequences at the C-terminus of cFLIP-L, which regulate the of cFLIP-L, at least in our system. These observations suggest that nuclear and cytoplasmic distribution of cFLIP-L. cFLIP-L has a the C-terminal NLS and NES in cFLIP-L regulate nuclear or tandem death effector domain (DED) at the N-terminus, and cytoplasmic localization independently of other DED-containing interacts with other DED-containing proteins such as FADD and proteins. caspase-8 in the death-inducing signaling complex (DISC). FADD We, and others, previously reported that cFLIP-L increases - and caspase-8 were originally reported to be cytoplasmic proteins, catenin accumulation and enhances Wnt signaling (Naito et al., but FADD contains an NLS sequence and shuttles between the 2004; Nakagiri et al., 2005). In A549 lung carcinoma cells, nuclear and cytoplasmic compartments (Gomez-Angelats and downregulation of the endogenous cFLIP protein results in reduced Cidlowski, 2003). The presence of caspase-8 in nuclei has also been Wnt signaling, suggesting a significant role of endogenous cFLIP- reported (Qin et al., 2001). Thus, the DED-containing proteins L in the modulation of Wnt signaling. This activity is due, at least incorporated into DISC localize in the nucleus, which could affect in part, to the inhibition of -catenin ubiquitylation. The cFLIP-L the nuclear localization of cFLIP-L. However, we do not think these protein is prone to aggregate in cells and impairs the ubiquitin- proteins target cFLIP-L to the nucleus by homophylic interaction proteasome system (UPS), thereby inhibiting the ubiquitylation of Nuclear cFLIP-L modulates Wnt 27

The C-terminal sequences of cFLIP-L evidently have an important role in the upregulation of endogenous -catenin and Wnt signaling, because deletion of the C-terminal sequences of cFLIP- L [FLIP(1-438)], and mutations in NLS or NES at the C-terminus of cFLIP-L (435mt, 472mt, 439mt) resulted in the abrogation of these activities (supplementary material Figs S2-S4). However, by conjugating the NES sequence from MAPKK to cFLIP-L, we successfully differentiated upregulation of -catenin and Wnt signaling modulation, by cFLIP-L containing an intact C-terminus. NES-cFLIP-L predominantly localized in the cytoplasm (Fig. 4B) and increased -catenin accumulation to levels that were similar to those in cells expressing wild-type cFLIP-L (Fig. 6A). Wnt3a- induced nuclear translocation of -catenin occurs equally in cells transfected with mock, wild-type cFLIP-L and NES-cFLIP-L (supplementary material Fig. S6). However, wild-type cFLIP-L Fig. 6. NES-cFLIP-L increases -catenin expression, but its activity to enhance -catenin-mediated gene expression is attenuated. (A,B)Plasmids markedly increased -catenin-mediated gene expression, whereas encoding wild-type cFLIP-L and NES-cFLIP-L (1.0g) were transfected into NES-cFLIP-L showed a seriously reduced activity (Fig. 5D, Fig. HEK293T cells with a TOP luciferase reporter plasmid (0.2g) and a control 6B). These results suggest a role for nuclear cFLIP-L in the reporter plasmid (pRL-TK, 0.02g). After 24 hours, cell lysates were modulation of Wnt signaling. In the nucleus, cFLIP-L interacts with analyzed by western blotting with the indicated antibodies (A), and the the TOP-TK-Luc reporter plasmid (Fig. 7), suggesting that cFLIP- luciferase activity was measured and expressed as the fold increase compared L directly associates with a transcriptional complex in -catenin- with the level observed in cells transfected without cFLIP (mock, B). mediated gene expression. Thus, cFLIP-L modulates Wnt signaling by two mechanisms: (1) in the cytoplasm, it regulates -catenin accumulation by impairing UPS, and (2) in the nucleus, cFLIP-L many proteins, including -catenin, resulting in an accumulation modulates Wnt signaling by directly associating with the of -catenin (Ishioka et al., 2007). However, -catenin accumulation transcriptional complex. Both of these activities and in the cells does not always result in the activation of -catenin- nucleocytoplasmic shuttling require the caspase-like domain, which mediated gene expression. For example, co-expression of -catenin distinguishes cFLIP-L from cFLIP-S in the modulation of Wnt with cFLIP-S [or cFLIP (1-438)] increased the cellular level of - signaling. catenin, as did co-expression with cFLIP-L. At the same time, -catenin-mediated gene expression was not induced by the co- Materials and Methods expression of cFLIP-S [or cFLIP(1-438)], whereas it was Cell culture conditions Human embryonic kidney 293T cells were cultured in Dulbecco’s modified Eagle’s remarkably induced by the co-expression of cFLIP-L medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (supplementary material Fig. S7). These results strongly suggest (FBS). Human fibrosarcoma HT1080, human lung cancer A549, human renal cell

Journal of Cell Science that cFLIP-L has an additional mechanism, other than the carcinoma ACHN and human prostate cancer DU145 cells were cultured in RPMI upregulation of -catenin accumulation, by which it modulates Wnt 1640 medium (RPMI 1640; Nissui, Tokyo, Japan) supplemented with 10% FBS. To  assess cell growth, the MTS assay was used. In brief, cells were incubated with a signaling. tetrazolium compound, MTS (Promega, Madison, WI; G3580), for 1 hour. The optical density was measured at 490 nm with a reference at 690 nm, using a microplate spectrophotometer (Benchmark Plus, Bio-Rad, Hercules, CA).

Plasmids Human cFLIP-L was cloned into pcDNA-based mammalian expression vectors as described previously (Naito et al., 2004). The EGFP-cFLIP variant expression plasmids were constructed by inserting cDNAs encoding cFLIP variants into EcoRI and SalI sites of the pEGFP-C2. Mutants of cFLIP were generated using a PCR-based mutagenesis kit (QuikChange site-directed mutagenesis kit, Stratagene). The NES- cFLIP-L expression plasmid was constructed by inserting the synthetic oligonucleotide encoding nuclear export signal (NES) of MAPKK into the EcoRI site of pcDNA3Myc- cFLIP-L. All of the constructs generated from PCR products were sequenced.

Transfection and immunoblotting HT1080 cells and HEK293T cells were transfected with various plasmid DNAs by lipofection [FuGENE6 (Roche); LipofectAMINE2000 (Invitrogen)]. In some cases, cells were treated with benzyloxycarbonyl-valinyl-alanyl-aspartate-fluoromethyl ketone (Z-VAD-fmk) (20 M) to inhibit the apoptosis induced by cFLIP-L overexpression. To prepare whole-cell lysates, cells were lysed in SDS lysis buffer Fig. 7. cFLIP-L physically interacts with the TOP reporter plasmid. (0.1 M Tris-HCl at pH 7.5, 10% glycerol, 1% SDS) for 5 minutes at 100°C, and cleared by centrifugation at 17,400 g for 10 minutes. For subcellular fractionation, HEK293T cells were transfected with various combinations of plasmids cells were extracted with NE-PER nuclear and cytoplasmic extraction reagent encoding wild-type or C-terminally deleted (1-438) cFLIP-L (16g) together (PIERCE). We used the following antibodies: anti-FLIP (NF-6 or Dave-II; Alexis with the TOP luciferase reporter plasmid (T, 4g) or control FOP reporter Biochemicals); anti--tubulin from Cosmobio; polyclonal anti-Myc from MBL; anti- plasmid (F, 4g). After 24 hours, cells were crosslinked with 1% hemagglutinin (anti-HA) and monoclonal anti-Myc from Roche; anti- from Santa formaldehyde and then lysed with lysis buffer. The lysates were sonicated and Cruz Biotechnology; anti-Topo-II from BD Biosciences transduction; and anti-FLAG diluted. Then, the lysates were incubated with an anti-FLIP antibody (Dave-II) (M2; Sigma). or anti-RNA polymerase II antibody, and the subsequent steps were performed Isolation of transfectant clones using an EZ ChIP chromatin immunoprecipitation kit. The isolated DNA was HT1080 cells were transfected with pcDNA3-cFLIP variants or pEGFP-cFLIP variants. amplified by PCR using the primers for reporter vectors or GAPDH. At 24 hours after transfection, the cells were selected with 600 g/ml G418 for 2 weeks, 28 Journal of Cell Science 123 (1)

and the surviving cells were cloned by limiting dilution. The transfectants were Fornerod, M., Ohno, M., Yoshida, M. and Mattaj, I. W. (1997). CRM1 is an export maintained in RPMI-1640 medium containing 10% heat-inactivated FBS, 100 g/ml receptor for leucine-rich nuclear export signals. Cell 90, 1051-1060. kanamycin and 200 g/ml G418 at 37°C in a humidified atmosphere of 5% CO2. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M. and Nishida, E. (1997). CRM1 is responsible for intracellular transport mediated by the Immunofluorescence microscopy nuclear export signal. Nature 390, 308-311. HT1080 or A549 cells were fixed in 4% paraformaldehyde for 15 minutes at room Gama-Carvalho, M. and Carmo-Fonseca, M. (2001). The rules and roles of temperature and treated with 0.1% Triton X-100, 3% bovine serum albumin in nucleocytoplasmic shuttling proteins. FEBS Lett. 498, 157-163. phosphate-buffered saline for 30 minutes at room temperature. Cells were incubated Gomez-Angelats, M. and Cidlowski, J. A. (2003). Molecular evidence for the nuclear with anti-FLIP (NF6, 1: 100) as the primary antibody overnight at 4°C and Alexa- localization of FADD. Cell Death Differ. 10, 791-797. Harel, A. and Forbes, D. J. (2004). Importin beta: conducting a much larger cellular Fluor-488-conjugated or Alexa-Fluor-568-conjugated anti-mouse IgG (1:1000) as the symphony. Mol. Cell 16, 319-330. secondary antibody for 1 hour at room temperature. The cells were observed with an Hood, J. K. and Silver, P. A. (1999). In or out? Regulating . Curr. Opin. Olympus FV1000 confocal microscope equipped with a charge-coupled device camera. Cell Biol. 11, 241-247. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, Luciferase assay J. L., Schroter, M., Burns, K., Mattmann, C. et al. (1997). Inhibition of death receptor Cells were transfected with various combinations of plasmids; 0.2 g reporter plasmid signals by cellular FLIP. Nature 388, 190-195. [TOP-TK-Luc, FOP-TK-Luc (Korinek et al., 1997)] or pNFB-Luc (Clontech), 0.02 Ishioka, T., Katayama, R., Kikuchi, R., Nishimoto, M., Takada, S., Takada, R., g internal control (pRL-TK; Clontech), 1 g cFLIP-L variants expression vector Matsuzawa, S., Reed, J. C., Tsuruo, T. and Naito, M. (2007). Impairment of the (pcDNA3-Myc) or empty pcDNA3 vector. In some cases, conditioned medium ubiquitin-proteasome system by cellular FLIP. Genes Cells 12, 735-744. containing Wnt3a, which was prepared from L-cells that had been transfected with Kataoka, T. and Tschopp, J. (2004). N-terminal fragment of c-FLIP(L) processed by the gene encoding Wnt3a as described previously (Shibamoto et al., 1998), was added caspase 8 specifically interacts with TRAF2 and induces activation of the NF-kappaB to the medium. Luciferase activities were measured at 24 hours after transfection, or signaling pathway. Mol. Cell. Biol. 24, 2627-2636. after treatment with the conditioned medium using the Dual-Luciferase Reporter Assay Kataoka, T., Budd, R. C., Holler, N., Thome, M., Martinon, F., Irmler, M., Burns, System (Promega). K., Hahne, M., Kennedy, N., Kovacsovics, M. et al. (2000). The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Curr. Biol. 10, DNA-protein interaction assay 640-648. HEK293T cells in 100 mm plates were transfected with a total of 20 g of various Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B. and Clevers, H. (1997). Constitutive transcriptional activation by a beta- combinations of plasmids; 4 g reporter plasmid (TOP-TK-Luc, FOP-TK-Luc), 16 catenin-Tcf complex in APC–/– colon carcinoma. Science 275, 1784-1787. g cFLIP-L variants expression vector (pcDNA3-Myc) or empty pcDNA3 vector.  Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E. P., Yoneda, Y., Yanagida, M., After transfection for 24 hours, cells were crosslinked for 10 minutes by directly Horinouchi, S. and Yoshida, M. (1998). Leptomycin B inhibition of signal-mediated adding 1% formaldehyde to the culture medium and then lysing them with lysis buffer nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540-547. (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1). The lysates were sonicated Kudo, N., Matsumori, N., Taoka, H., Fujiwara, D., Schreiner, E. P., Wolff, B., Yoshida, seven times for 10 seconds with output level 4 (Sonifier, Branson Ultrasonic). For M. and Horinouchi, S. (1999). Leptomycin B inactivates CRM1/exportin 1 by covalent chromatin immunoprecipitation, the cell lysates were incubated with an anti-FLIP modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. antibody (Dave-II, Alexis) or anti-RNA polymerase II antibody (CTD4H8, Upstate, USA 96, 9112-9117. Charlottesville, VG), and the following steps were performed using an EZ ChIP Lange, A., Mills, R. E., Lange, C. J., Stewart, M., Devine, S. E. and Corbett, A. H. chromatin immunoprecipitation kit (Upstate). The isolated DNA was amplified by (2007). Classical nuclear localization signals: definition, function, and interaction with PCR using an EX Taq DNA polymerase (TaKaRa) with the following primers importin alpha. J. Biol. Chem. 282, 5101-5105. (GAPDH-F, 5Ј-TACTAGCGGTTTTACGGGCG-3Ј; GAPDH-R, 5Ј-TCGAACA - Logan, C. Y. and Nusse, R. (2004). The Wnt signaling pathway in development and disease. GGAGCAGAGAGCGA-3Ј; reporter-128F, 5Ј-GTGTCGGGGCTGGCT TA AC T - Annu. Rev. Cell Dev. Biol. 20, 781-810. ATGCGG-3Ј; reporter-593R; 5Ј-TCGGGCACGCTGTTGACGCTGTTAAG-3Ј). Mattaj, I. W. and Englmeier, L. (1998). Nucleocytoplasmic transport: the soluble phase. Annu. Rev. Biochem. 67, 265-306. Naito, M., Katayama, R., Ishioka, T., Suga, A., Takubo, K., Nanjo, M., Hashimoto, We thank Tetsu Akiyama (Institute for Molecular and Cellular C., Taira, M., Takada, S., Takada, R. et al. (2004). Cellular FLIP inhibits beta-catenin Biosciences, The University of Tokyo, Tokyo) for -catenin plasmid, ubiquitylation and enhances Wnt signaling. Mol. Cell. Biol. 24, 8418-8427. Hans Clevers (Hubrecht Institute, Utrecht, The Netherlands) for TOP- Nakagiri, S., Murakami, A., Takada, S., Akiyama, T. and Yonehara, S. (2005). Viral Journal of Cell Science TK-Luc and FOP-TK-Luc reporter plasmids. We also thank Akihiro FLIP enhances Wnt signaling downstream of stabilized beta-catenin, leading to control of cell growth. Mol. Cell. Biol. 25, 9249-9258. Tomida (Japanese Foundation for Cancer Research, Tokyo) for helpful Nakajima, A., Komazawa-Sakon, S., Takekawa, M., Sasazuki, T., Yeh, W. C., Yagita, discussions. This study was supported by Grants-in-Aid for Cancer H., Okumura, K. and Nakano, H. (2006). An antiapoptotic protein, c-FLIPL, directly Research and Scientific Research from the Ministry of Education, binds to MKK7 and inhibits the JNK pathway. EMBO J. 25, 5549-5559. Science, Sports and Culture of Japan. Pacific Edit reviewed the Nigg, E. A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779-787. manuscript prior to submission. Qin, Z. H., Wang, Y., Kikly, K. K., Sapp, E., Kegel, K. B., Aronin, N. and DiFiglia, Supplementary material available online at M. (2001). Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation. J. Biol. Chem. 276, 8079-8086. http://jcs.biologists.org/cgi/content/full/123/1/23/DC1 Schickling, O., Stegh, A. H., Byrd, J. and Peter, M. E. (2001). Nuclear localization of DEDD leads to caspase-6 activation through its death effector domain and inhibition of References RNA polymerase I dependent transcription. Cell Death Differ. 8, 1157-1168. Andersson, C. X., Fernandez-Rodriguez, J., Laos, S., Sikut, R., Sikut, A., Baeckstrom, Shibamoto, S., Higano, K., Takada, R., Ito, F., Takeichi, M. and Takada, S. (1998). D. and Hansson, G. C. (2004). CD43 has a functional NLS, interacts with beta-catenin, Cytoskeletal reorganization by soluble Wnt-3a protein signalling. Genes Cells 3, 659- and affects gene expression. Biochem. Biophys. Res. Commun. 316, 12-17. 670. Chaudhary, P. M., Eby, M. T., Jasmin, A., Kumar, A., Liu, L. and Hood, L. (2000). Stade, K., Ford, C. S., Guthrie, C. and Weis, K. (1997). Exportin 1 (Crm1p) is an essential Activation of the NF-kappaB pathway by caspase 8 and its homologs. Oncogene 19, nuclear export factor. Cell 90, 1041-1050. 4451-4460. Wang, Y., Kakinuma, N., Zhu, Y. and Kiyama, R. (2006). Nucleo-cytoplasmic shuttling Chung, J. H. and Eng, C. (2005). Nuclear-cytoplasmic partitioning of phosphatase and of human Kank protein accompanies intracellular translocation of beta-catenin. J. Cell tensin homologue deleted on chromosome 10 (PTEN) differentially regulates the cell Sci. 119, 4002-4010. cycle and apoptosis. Cancer Res. 65, 8096-8100. Weis, K. (1998). Importins and exportins: how to get in and out of the nucleus. Trends Clevers, H. (2006). Wnt/beta-catenin signaling in development and disease. Cell 127, 469- Biochem. Sci. 23, 185-189. 480. Wen, W., Meinkoth, J. L., Tsien, R. Y. and Taylor, S. S. (1995). Identification of a signal Fang, L. W., Tai, T. S., Yu, W. N., Liao, F. and Lai, M. Z. (2004). Phosphatidylinositide for rapid export of proteins from the nucleus. Cell 82, 463-473. 3-kinase priming couples c-FLIP to T cell activation. J. Biol. Chem. 279, 13-18. Wolff, B., Sanglier, J. J. and Wang, Y. (1997). Leptomycin B is an inhibitor of nuclear Fasken, M. B., Saunders, R., Rosenberg, M. and Brighty, D. W. (2000). A leptomycin export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency B-sensitive homologue of human CRM1 promotes nuclear export of nuclear export virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4, 139-147. sequence-containing proteins in Drosophila cells. J. Biol. Chem. 275, 1878-1886. Zhan, Y., Hegde, R., Srinivasula, S. M., Fernandes-Alnemri, T. and Alnemri, E. S. Fischer, U., Huber, J., Boelens, W. C., Mattaj, I. W. and Luhrmann, R. (1995). The (2002). Death effector domain-containing proteins DEDD and FLAME-3 form nuclear HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway complexes with the TFIIIC102 subunit of human transcription factor IIIC. Cell Death used by specific cellular . Cell 82, 475-483. Differ. 9, 439-447.