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 β-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 β-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.
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-κB, 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 β-catenin, which is a mediator of canonical Wnt signaling (Naito et al., 2004; Nakagiri et al., 2005). In unstimulated cells, free cytosolic β-catenin is maintained at a low level by serine or threonine phosphorylation of β-catenin, followed by ubiquitylation and degradation by the proteasome. When cells are stimulated with Wnt3a, the phosphorylation of β-catenin is inhibited, resulting in the accumulation and translocation of β-catenin into the nucleus. In the nucleus, β-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 β-catenin, resulting in an enhancement of Wnt signaling (Ishioka et al., 2007; Naito et al., 2004).
In this study, we show that cFLIP-L has NLS and NES sequences at the C-terminus, and shuttles between the nucleus and the cytoplasm. Forced expression of cFLIP-L in the cytoplasm by conjugation of NES to cFLIP-L abrogated the ability of cFLIP-L to enhance Wnt signaling, while retaining the capacity to increase the β-catenin protein level. In addition, cFLIP-L physically interacted with the reporter plasmid for Wnt signaling. These results suggest that nuclear cFLIP-L has a role in modulating Wnt signaling.
Nuclear and cytoplasmic localization of cFLIP-L
After ligation of death receptor, cFLIP-L is recruited to the death-inducing signaling complex (DISC) composed of death receptor, FADD and caspase-8, and therefore, cFLIP-L is regarded as a cytoplasmic protein. However, when cells constitutively expressing Myc-tagged cFLIP-L were stained with anti-Myc antibody, a strong signal was observed in nuclei (Fig. 1A). Biochemical fractionation also showed that a significant amount of Myc-tagged cFLIP-L was extracted in the nuclear fraction (Fig. 1B). We also examined the localization of endogenous cFLIP protein by immunocytochemical staining in A549 cells, and found cFLIP protein distributed in the cytoplasm and nuclei (Fig. 1C). Cytoplasmic and nuclear 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 indicate that cFLIP-L localizes in both the nucleus and cytoplasm, whereas cFLIP-S is exclusively found in the cytoplasm.
Leptomycin B increases nuclear localization of cFLIP-L
It is known that many proteins localizing in both the nucleus and cytoplasm shuttle between the two compartments. The transport from the nucleus to the cytoplasm is often mediated by CRM1 (Fornerod et al., 1997; Gama-Carvalho and Carmo-Fonseca, 2001; Harel and Forbes, 2004; Stade et al., 1997), which can be inhibited specifically by an antibiotic, leptomycin B (LMB) (Fasken et al., 2000; Kudo et al., 1999; Kudo et al., 1998; Wolff et al., 1997). To examine whether cFLIP-L is exported in a CRM1-dependent manner, we generated HT1080 cells constitutively expressing EGFP-cFLIP-L, and examined the localization of the EGFP-cFLIP-L with or without LMB. The EGFP-cFLIP-L exhibited cytoplasmic and nuclear localization without LMB treatment, and the accumulation of the protein in nuclei was increased after treatment with LMB (Fig. 2A). Nuclear and cytoplasmic fractionation of the cells confirmed the nuclear accumulation of cFLIP-L (∼1.6-fold increase) by LMB treatment (Fig. 2B). On the other hand, EGFP-cFLIP-S protein localizes abundantly in the cytoplasm, and the pattern of distribution did not change after LMB treatment (Fig. 2B). We also examined the localization of endogenous cFLIP-L in A549 cells treated with LMB. As shown in Fig. 2C, LMB treatment increased the nuclear accumulation of endogenous cFLIP-L. Collectively, these results suggest that cFLIP-L, but not cFLIP-S, shuttles between the cytoplasm and the nucleus.
Identification of the NLS and NES sequences in cFLIP-L
Because cFLIP-L, but not cFLIP-S or a cFLIP-L mutant lacking 42 amino acids of the C-terminal [cFLIP(1-438)], localized in the nucleus (Figs 1, 2 and supplementary material Fig. S1), we reasoned that NLS might be present at the C-terminus of cFLIP-L. We searched for the NLS-like sequence at the C-terminal region of cFLIP-L, and found a pair of three consecutive basic amino acids residues (435RKR and 472RKK) separated by 34 amino acids that could potentially function as an NLS (Fig. 3A, red). We also found an NES-like sequence near the putative NLS in cFLIP-L (Fig. 3A, green). Then we examined whether the NLS- and NES-like sequences regulate the nuclear and cytoplasmic localization of cFLIP-L by mutation analysis. We generated cells constitutively expressing NLS-mutants (435mt and 472mt) and an NES-mutant (439mt) of cFLIP-L (Fig. 3A), and examined the protein localization. The NLS mutant cFLIP-L proteins predominantly localized in the cytoplasm, whereas the NES mutant localized in the nucleus (Fig. 3B). These results indicate that the localization of cFLIP-L is regulated by the NLS and NES sequences at the C-terminus of cFLIP-L.
Mutations in the C-terminus of cFLIP-L abrogate the activity to modulate Wnt signaling
As the subcellular localization of a protein is closely related to its function (Andersson et al., 2004; Chung and Eng, 2005; Gama-Carvalho and Carmo-Fonseca, 2001; Gomez-Angelats and Cidlowski, 2003; Schickling et al., 2001; Wang et al., 2006; Zhan et al., 2002), we hypothesized that the NLS and NES mutants of cFLIP-L might have a different function from wild-type cFLIP-L. Cells constitutively expressing the wild-type and mutant cFLIP-L proteins (supplementary material Fig. S2A) displayed resistance to the apoptosis induced by an anti-Fas antibody (supplementary material Fig. S2B), but not by an anti-tumor drug, etoposide (supplementary material Fig. S2C), suggesting that anti-apoptotic function of the NLS- and NES-mutant cFLIP-L proteins is indistinguishable from that of the wild-type cFLIP-L. We next treated the cells with Wnt3a and measured β-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 β-catenin, and induced β-catenin-mediated gene expression, whereas these activities were completely abolished in 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 β-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 sensitive to the anti-tumor drug, etoposide (Fig. 5C). We observed no differences in NF-κB activation in cells expressing wild-type, NLS mutant or NES-cFLIP-L (supplementary material Fig. S5). However, Wnt3a-induced gene expression was enhanced in the wild-type cFLIP-L transfectants, but not in the NES-cFLIP-L transfectants (Fig. 5D), although nuclear translocation of β-catenin by Wnt3a treatment was equally observed in these cells (supplementary material Fig. S6). Transient transfection of wild-type cFLIP-L and NES-cFLIP-L equally increased β-catenin accumulation (Fig. 6A), but the β-catenin-mediated gene expression was greatly reduced in the NES-cFLIP-L-expressing cells compared 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 cFLIP-L that is essential for FLIP-dependent Wnt pathway activation, these results suggest that cFLIP-L activates the Wnt signaling pathway through a mechanism independently of β-catenin stabilization or downstream of the stabilization, which could take place in the nucleus.
To study further the role of the nuclear cFLIP-L in Wnt signaling modulation, we examined whether cFLIP-L physically interacts with the reporter plasmid for β-catenin-mediated gene expression. Fig. 7 shows that immunoprecipitates of cFLIP-L contained the reporter plasmid TOP-TK-Luc, but not the control plasmid FOP-TK-Luc. cFLIP-L(1-438), which does not enhance Wnt signaling, was devoid of this activity. These results suggest a direct role for nuclear cFLIP-L in the modulation of β-catenin-mediated gene expression.
Nucleocytoplasmic transportation of proteins has an important role in the regulation of many cellular processes. Previous studies reported that cFLIP-L is a cytoplasmic protein that inhibits the apoptosis signaling initiated by death receptor ligation at the cell membrane. In this study, we report for the first time that cFLIP-L localizes in the nucleus and cytoplasm, whereas cFLIP-S predominantly localizes in the cytoplasm. We identified NLS and NES sequences at the C-terminus of cFLIP-L, which regulate the nuclear and cytoplasmic distribution of cFLIP-L. cFLIP-L has a tandem death effector domain (DED) at the N-terminus, and interacts with other DED-containing proteins such as FADD and caspase-8 in the death-inducing signaling complex (DISC). FADD and caspase-8 were originally reported to be cytoplasmic proteins, but FADD contains an NLS sequence and shuttles between the nuclear and cytoplasmic compartments (Gomez-Angelats and Cidlowski, 2003). The presence of caspase-8 in nuclei has also been reported (Qin et al., 2001). Thus, the DED-containing proteins incorporated into DISC localize in the nucleus, which could affect the nuclear localization of cFLIP-L. However, we do not think these proteins target cFLIP-L to the nucleus by homophylic interaction with DED, because cFLIP-S, which also contains DED and is recruited to DISC, did not localize in the nucleus (Fig. 1D). cFLIP-L also interacts with other DED-containing proteins, such as DEDD1 and DEDD2, which are nuclear proteins that sequester cFLIP in the nucleus upon overexpression in human cell lines (Zhan et al., 2002). However, DEDD2 overexpression sequestered the cFLIP protein in nucleoli, whereas the wild-type and NES-mutant cFLIP-L proteins localized in the nucleoplasm (Figs 1 and 2), suggesting that DEDD2 is not involved in the nuclear localization of cFLIP-L, at least in our system. These observations suggest that the C-terminal NLS and NES in cFLIP-L regulate nuclear or cytoplasmic localization independently of other DED-containing proteins.
We, and others, previously reported that cFLIP-L increases β-catenin accumulation and enhances Wnt signaling (Naito et al., 2004; Nakagiri et al., 2005). In A549 lung carcinoma cells, downregulation of the endogenous cFLIP protein results in reduced Wnt signaling, suggesting a significant role of endogenous cFLIP-L in the modulation of Wnt signaling. This activity is due, at least in part, to the inhibition of β-catenin ubiquitylation. The cFLIP-L protein is prone to aggregate in cells and impairs the ubiquitin-proteasome system (UPS), thereby inhibiting the ubiquitylation of many proteins, including β-catenin, resulting in an accumulation of β-catenin (Ishioka et al., 2007). However, β-catenin accumulation in the cells does not always result in the activation of β-catenin-mediated gene expression. For example, co-expression of β-catenin with cFLIP-S [or cFLIP (1-438)] increased the cellular level of β-catenin, as did co-expression with cFLIP-L. At the same time, β-catenin-mediated gene expression was not induced by the co-expression of cFLIP-S [or cFLIP(1-438)], whereas it was remarkably induced by the co-expression of cFLIP-L (supplementary material Fig. S7). These results strongly suggest that cFLIP-L has an additional mechanism, other than the upregulation of β-catenin accumulation, by which it modulates Wnt signaling.
The C-terminal sequences of cFLIP-L evidently have an important role in the upregulation of endogenous β-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 β-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 β-catenin accumulation to levels that were similar to those in cells expressing wild-type cFLIP-L (Fig. 6A). Wnt3a-induced nuclear translocation of β-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 markedly increased β-catenin-mediated gene expression, whereas NES-cFLIP-L showed a seriously reduced activity (Fig. 5D, Fig. 6B). These results suggest a role for nuclear cFLIP-L in the modulation of Wnt signaling. In the nucleus, cFLIP-L interacts with the TOP-TK-Luc reporter plasmid (Fig. 7), suggesting that cFLIP-L directly associates with a transcriptional complex in β-catenin-mediated gene expression. Thus, cFLIP-L modulates Wnt signaling by two mechanisms: (1) in the cytoplasm, it regulates β-catenin accumulation by impairing UPS, and (2) in the nucleus, cFLIP-L modulates Wnt signaling by directly associating with the transcriptional complex. Both of these activities and nucleocytoplasmic shuttling require the caspase-like domain, which distinguishes cFLIP-L from cFLIP-S in the modulation of Wnt signaling.
Materials and Methods
Cell culture conditions
Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS). Human fibrosarcoma HT1080, human lung cancer A549, human renal cell carcinoma ACHN and human prostate cancer DU145 cells were cultured in RPMI 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 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).
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 μM) to inhibit the apoptosis induced by cFLIP-L overexpression. To prepare whole-cell lysates, cells were lysed in SDS lysis buffer (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, cells were extracted with NE-PER nuclear and cytoplasmic extraction reagent (PIERCE). We used the following antibodies: anti-FLIP (NF-6 or Dave-II; Alexis Biochemicals); anti-α-tubulin from Cosmobio; polyclonal anti-Myc from MBL; anti-hemagglutinin (anti-HA) and monoclonal anti-Myc from Roche; anti-actin from Santa Cruz Biotechnology; anti-Topo-IIβ from BD Biosciences transduction; and anti-FLAG (M2; Sigma).
Isolation of transfectant clones
HT1080 cells were transfected with pcDNA3-cFLIP variants or pEGFP-cFLIP variants. At 24 hours after transfection, the cells were selected with 600 μg/ml G418 for 2 weeks, and the surviving cells were cloned by limiting dilution. The transfectants were maintained in RPMI-1640 medium containing 10% heat-inactivated FBS, 100 μg/ml kanamycin and 200 μg/ml G418 at 37°C in a humidified atmosphere of 5% CO2.
HT1080 or A549 cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature and treated with 0.1% Triton X-100, 3% bovine serum albumin in phosphate-buffered saline for 30 minutes at room temperature. Cells were incubated with anti-FLIP (NF6, 1: 100) as the primary antibody overnight at 4°C and Alexa-Fluor-488-conjugated or Alexa-Fluor-568-conjugated anti-mouse IgG (1:1000) as the secondary antibody for 1 hour at room temperature. The cells were observed with an Olympus FV1000 confocal microscope equipped with a charge-coupled device camera.
Cells were transfected with various combinations of plasmids; 0.2 μg reporter plasmid [TOP-TK-Luc, FOP-TK-Luc (Korinek et al., 1997)] or pNFκB-Luc (Clontech), 0.02 μg internal control (pRL-TK; Clontech), 1 μg cFLIP-L variants expression vector (pcDNA3-Myc) or empty pcDNA3 vector. In some cases, conditioned medium containing Wnt3a, which was prepared from L-cells that had been transfected with the gene encoding Wnt3a as described previously (Shibamoto et al., 1998), was added to the medium. Luciferase activities were measured at 24 hours after transfection, or after treatment with the conditioned medium using the Dual-Luciferase Reporter Assay System (Promega).
DNA-protein interaction assay
HEK293T cells in 100 mm plates were transfected with a total of 20 μg of various combinations of plasmids; 4 μg reporter plasmid (TOP-TK-Luc, FOP-TK-Luc), 16 μg cFLIP-L variants expression vector (pcDNA3-Myc) or empty pcDNA3 vector. After transfection for 24 hours, cells were crosslinked for 10 minutes by directly adding 1% formaldehyde to the culture medium and then lysing them with lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1). The lysates were sonicated seven times for 10 seconds with output level 4 (Sonifier, Branson Ultrasonic). For chromatin immunoprecipitation, the cell lysates were incubated with an anti-FLIP antibody (Dave-II, Alexis) or anti-RNA polymerase II antibody (CTD4H8, Upstate, Charlottesville, VG), and the following steps were performed using an EZ ChIP chromatin immunoprecipitation kit (Upstate). The isolated DNA was amplified by PCR using an EX Taq DNA polymerase (TaKaRa) with the following primers (GAPDH-F, 5′-TACTAGCGGTTTTACGGGCG-3′; GAPDH-R, 5′-TCGAACAGGAGCAGAGAGCGA-3′; reporter-128F, 5′-GTGTCGGGGCTGGCTTAACTATGCGG-3′; reporter-593R; 5′-TCGGGCACGCTGTTGACGCTGTTAAG-3′).
We thank Tetsu Akiyama (Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo) for β-catenin plasmid, Hans Clevers (Hubrecht Institute, Utrecht, The Netherlands) for TOP-TK-Luc and FOP-TK-Luc reporter plasmids. We also thank Akihiro Tomida (Japanese Foundation for Cancer Research, Tokyo) for helpful discussions. This study was supported by Grants-in-Aid for Cancer Research and Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Pacific Edit reviewed the manuscript prior to submission.