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 Chemotherapy 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 apoptosis 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 nuclear export signal (NES) near the NLS, and leptomycin 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 proteins 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 protein, 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 leucine 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 phosphorylation 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 cysteine 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 antibiotic, 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 antibody, 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: 20m. (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 antibodies. 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: 20m. (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 20g protein from each fraction was analyzed by western the cFLIP band in each fraction (average of two experiments) compared with blotting with the indicated antibodies.