23 Research Article Introduction Nucleocytoplasmic shuttling of proteins has an important role in cell function (Weis, 1998). The coordination of trafficking between the nucleus and cytoplasm is determined by the balance of nuclear import and export activity (Hood and Silver, 1999; Mattaj and Englmeier, 1998; Nigg, 1997). Proteins destined for transport into the nucleus contain amino acid targeting sequences called nuclear localization signals (NLSs), which principally consist of either one (monopartite) or two (bipartite) stretches of basic amino acids (Lange et al., 2007). By contrast, proteins that are exported from the nucleus mainly contain nuclear export signal (NES) sequences, which consist of several leucine residues distributed with an uneven spacing (Fischer et al., 1995; Wen et al., 1995). A related family of shuttling transport factors, the importins and exportins, recognize the NLS- or NES-containing proteins and mediate nuclear import or export, respectively. Leptomycin B (LMB) effectively blocks protein export from the nucleus by directly binding to exportin-1 (CRM1) (Fukuda et al., 1997). Cellular FLIP (cFLIP, also known as I-FLICE, FLAME1, Casper, CASH, MRIT and Usurpin) is an inhibitor of the apoptosis initiated by death receptor ligation (Irmler et al., 1997). The long form of c-FLIP (cFLIP-L) is highly homologous to caspase-8, and contains two death effector domains (DED) and a caspase-like domain at the N- and C-termini, respectively. cFLIP-L, however, does not have caspase activity because it has no 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. Previous studies revealed the presence of a short isoform of FLIP protein, cFLIP-S, and both isoforms of cFLIP can inhibit death-receptor-mediated apoptosis. In addition to apoptosis inhibition, cFLIP-L mediates the activation of NF-B, PI3K and Erk by virtue of its capacity to recruit the adaptor proteins involved in each signaling pathway, such as TRAF-1, TRAF-2, RIP and Raf-1 (Chaudhary et al., 2000; Fang et al., 2004; Kataoka et al., 2000; Kataoka and Tschopp, 2004). Inhibition of the JNK pathway by direct binding to MKK7 has also been reported (Nakajima et al., 2006). All of these phenomena take place in the cytoplasm, and therefore, cFLIP is regarded as a cytoplasmic protein. 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. When cells are stimulated with Wnt3a, the phosphorylation of -catenin is inhibited, resulting in the accumulation and translocation of -catenin into the nucleus. In the nucleus, -catenin interacts with T-cell-factor/lymphoid-enhancing factor (TCF/LEF), and activates their target genes to promote cell growth (Clevers, 2006; Logan and Nusse, 2004). Exogenously expressed and endogenous cFLIP-L expressed in some cancer cells are prone to aggregate in the cells and inhibit the ubiquitylation of short-lived proteins, including -catenin, resulting in an enhancement of Wnt signaling (Ishioka et al., 2007; Naito et al., 2004). Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L Ryohei Katayama 1,2 , Toshiyasu Ishioka 1 , Shinji Takada 3 , Ritsuko Takada 3 , Naoya Fujita 2 , Takashi Tsuruo 2 and Mikihiko Naito 1,4, * 1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan 2 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan 3 Okazaki Institute for Integrative Biosciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan 4 National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan *Author for correspondence (miki-naito@nihs.go.jp) 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 Journal of Cell Science Journal of Cell Science