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1 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
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Tokyo R & D Center, Daiichi Pharmaceutical Co., Ltd, Tokyo 134-8630, Japan
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Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Tokyo 135-8550, Japan
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BioResource Center, RIKEN, Tsukuba, Ibaragi 305-0074, Japan
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Abstract |
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Introduction |
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c-Ski is a nuclear oncoprotein originally identified as a cellular counterpart of a retroviral oncogene product, v-Ski (Li et al. 1986). c-Ski and SnoN, homologous proteins with similar function, together constitute a gene family. Over-expression of c-Ski as well as v-Ski induces morphologic transformation, anchorage independence, and myogenic differentiation of chicken and quail embryonic cells (Colmenares et al. 1991). Subsequently, c-Ski was found to physically interact with Smad2, Smad3, and Smad4 to antagonize signal transduction in the TGF-ß pathway (Akiyoshi et al. 1999; Luo et al. 1999; Sun et al. 1999; Xu et al. 2000). Although BMP-specific R-Smads bind to c-Ski only weakly (Akiyoshi et al. 1999; Mizuide et al. 2003), it has been shown that c-Ski inhibits BMP signaling in mammals as well as in Xenopus (Amaravadi et al. 1997; Wang et al. 2000) through interaction with Smad4 (Takeda et al. 2004). Over-expression of c-Ski abolishes TGF-ß-induced growth inhibition (Luo et al. 1999; Sun et al. 1999; Xu et al. 2000; Suzuki et al. 2004). High levels of expression of c-Ski have been reported in several tumor cell lines (Nomura et al. 1989; Fumagalli et al. 1993; Fukuchi et al. 2004) as well as in melanomas in vivo (Reed et al. 2001). Inhibition of TGF-ß signaling is thus considered to be a part of the mechanism of oncogenesis due to c-Ski (He et al. 2003).
c-Ski-mediated repression of TGF-ß superfamily signaling has been principally attributed to modulation of transcription (Liu et al. 2001) through recruitment of the nuclear corepressor (N-CoR) and histone deacetylase to Smad complexes as well as interference with recruitment of the transcriptional coactivator, p300/CBP (Akiyoshi et al. 1999; Wu et al. 2002). Recent structural analysis of the c-Ski-Smad4 complex suggested an alternative model in which c-Ski inactivates the R-Smad-Co-Smad complex through inhibition of correct complex formation (Wu et al. 2002), although direct physical interaction between R-Smads and Smad4 is not disrupted by c-Ski (Ueki & Hayman 2003; Takeda et al. 2004). We recently found that c-Ski enhances the binding of Smad complex to DNA, and suggested the possibility that inactive Smad complex is stabilized on the promoter regions of target genes by c-Ski (Suzuki et al. 2004).
Although c-Ski has been reported to be predominantly localized in the nucleus (Colmenares et al. 1991), recent reports have described cytoplasmic localization of c-Ski in some tumor cells including metastatic melanomas (Reed et al. 2001) and esophageal squamous cell carcinoma (Fukuchi et al. 2004). A role for cytoplasmic c-Ski in the inhibition of TGF-ß superfamily signaling has also been proposed (Reed et al. 2001; Kokura et al. 2003; Prunier et al. 2003).
In the present study, we identified a nuclear localization signal (NLS) in c-Ski, and introduced a mutation to abolish its function. Using the mutant, c-Ski (NLS mut), we found that nuclear and cytoplasmic c-Ski differently modulate cellular functions. We also observed that inhibition of proteasome activity causes accumulation of cytosplasmic c-Ski, suggesting that nuclear localization of c-Ski requires certain proteasome-regulated processes. Our findings suggest a novel mechanism regulating the activities of c-Ski by alteration of its subcellular localization.
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Results |
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We first searched for nuclear localization signals (NLSs) in the amino acid sequence of human c-Ski using PredictNLS Online (http://cubic.bioc.columbia.edu/cgi/var/nair/resonline.pl) and NucPred (http://sbcweb.pdc.kth.se/cgi-bin/maccallr/nucpred/single.pl), and found an amino acid sequence, PRKRKLT, corresponding to residues 452–458 in human c-Ski, as a candidate for NLS (Fig. 1A). A mutation to abolish the putative NLS function was then introduced by replacing lysine and arginine residues by asparagine and glutamine residues, respectively (NLS mut, Fig. 1A). The mutated c-Ski migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than wild-type c-Ski (c-Ski wt) did (Fig. 1B). Subcellular localization of c-Ski wt and the mutant was then examined in transfected HeLa cells (Fig. 1C), with distribution scored as nucleus (N), nucleus + cytoplasm (N + C), or cytoplasm (C) (Fig. 1D). c-Ski wt was predominantly localized in the nucleus (N, 88%; N + C, 8%; C, 4%). In contrast, mutant c-Ski was localized mainly in the cytoplasm (N, 1%; N + C, 65%; C, 34%). The sequence motif PRKRKLT in c-Ski thus promotes its nuclear localization.
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Cytoplasmic c-Ski is more stable than nuclear c-Ski
We next examined whether subcellular localization affects the stability of c-Ski. We compared the stabilities of c-Ski wt and c-Ski (NLS mut) by pulse-chase assay and found that c-Ski (NLS mut) is more stable than c-Ski wt (Fig. 2). We could not determine the differences in ubiquitination between c-Ski wt and c-Ski (NLS mut), because c-Ski wt was translocated to the cytoplasm in the presence of proteasome inhibitors, as described in succeeding discussions.
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We then examined whether c-Ski (NLS mut) affects TGF-ß and BMP signaling, by luciferase reporter assays (Fig. 3). c-Ski (NLS mut) inhibited TGF-ß-induced transactivation of (CAGA)9-MLP-Luc reporter as well as BMP-4-induced transactivation of BRE-Luc reporter (Fig. 3A,B).
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c-Ski has been found to enhance myogenic differentiation in vitro and in vivo (Sutrave et al. 1990; Colmenares et al. 1991), and has been reported to transactivate a promoter construct of myogenin (Ichikawa et al. 1997). As shown in Fig. 3D, c-Ski enhanced the activity of myogenin promoter construct MYO184-Luc in C2C12 cells, whereas c-Ski (NLS mut) did not. These findings further confirmed the lack of nuclear function of c-Ski (NLS mut).
Effects of c-Ski (NLS mut) on TGF-ß-induced cellular responses were then examined (Fig. 4). NMuMG mouse mammary epithelial cells were infected with lentivirus constructs encoding c-Ski wt or c-Ski (NLS mut), and their subcellular localization was confirmed in the absence (Fig. 4A) and in the presence of ligand stimulation (TGF-ß1 for 24 h, data not shown). Figure 4B shows the morphology of cells expressing green fluorescent protein (GFP), c-Ski wt, or c-Ski (NLS mut). Infected cells were visualized by the fluorescence of GFP, or internal ribosome entry site-linked Venus. c-Ski wt and c-Ski (NLS mut) each suppressed TGF-ß-induced epithelial-mesenchymal transdifferentiation (EMT) (Fig. 4B). Similarly, they abrogated TGF-ß-induced growth inhibition as determined by thymidine incorporation assay (Fig. 4C).
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c-Ski (NLS mut) requires interaction with either R-Smads or Co-Smad for inhibition of TGF-ß and BMP signaling
We next attempted to elucidate the mechanism of cytoplasmic c-Ski-mediated inhibition of TGF-ß/BMP signaling. As expected from its subcellular localization, c-Ski (NLS mut) appeared to suppress Smad signaling by a mechanism other than direct modulation of transcription. We previously found that interaction with Smad4 is indispensable for nuclear c-Ski-mediated suppression of BMP signaling but not for that of TGF-ß signaling (Takeda et al. 2004). To examine the requirement of Smad binding for cytoplasmic c-Ski-mediated suppression of TGF-ß/BMP signaling, we constructed a series of mutants combining NLS mutation with Smad binding-defective mutations, i.e. c-Ski (NLS mut)-
S2/3 and c-Ski (NLS mut)-W274E. We confirmed the cytoplasmic localization of these mutants and examined their effects on Smad signaling using luciferase reporter assays (Fig. 5). TGF-ß signaling was inhibited by c-Ski (NLS mut)-S2/3, which lacks interaction with Smad2/3, as well as by c-Ski (NLS mut)-W274E, which lacks interaction with Smad4, whereas BMP signaling was inhibited by c-Ski (NLS mut)-S2/3 but not by c-Ski (NLS mut)-W274E. We thus concluded that the inhibition of TGF-ß/BMP signaling by cytoplasmic c-Ski requires interaction with either R-Smad or Co-Smad.
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We further explored in detail how c-Ski (NLS mut) suppresses Smad functions. We first examined effects on R-Smad (Smad2 and Smad5) phosphorylation. c-Ski wt and c-Ski (NLS mut) each failed to inhibit phosphorylation of Smad2 induced by TGF-ß signaling, but rather enhanced it (Fig. 6A, left panel), whereas they did not affect phosphorylation of Smad5 induced by BMP signaling (Fig. 6A, right panel). These results suggest that c-Ski wt and c-Ski (NLS mut) suppress Smad function through the mechanism other than inhibition of R-Smad phosphorylation. We next examined whether c-Ski (NLS mut) affects the nuclear translocation of Smad proteins in response to signaling (Fig. 6B). We could not obtain clear results on the translocation of phospho-Smad2 in response to TGF-ß, because anti-phospho-Smad2 antibodies currently available were not applicable to immunocytochemistry. In transfected C2C12 cells, c-Ski (NLS mut) as well as c-Ski (NLS mut)-S2/3 suppressed the nuclear translocation of phospho-Smad1/5/8 induced by BMP, although they do not interact with Smad1/5/8. They thus appear to sequester R-Smad-Co-Smad complex through interaction with Smad4. On the other hand, c-Ski (NLS mut)–W274E, lacking interaction with Smad4 in addition to Smad1/5/8, failed to suppress the phospho-Smad1/5/8 translocation induced by BMP. The effects of c-Ski mutants on Smad translocation appear to be well correlated with their inhibition of Smad signaling (Fig. 5B). Supporting these results, we also observed that c-Ski (NLS mut) interacted with phospho-Smads in immunoprecipitation assays (Fig. 6C). We therefore conclude that cytoplasmic c-Ski suppresses Smad signaling through sequestration of R-Smad-Co-Smad complex in the cytoplasm.
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Because subcellular localization of c-Ski affects its activities in cells, we examined factors that affect subcellular localization of c-Ski. We found that treatment with MG132 or lactacystin caused cytoplasmic accumulation of c-Ski (Fig. 7A,B), suggesting a proteasome-dependent mechanism. In contrast, the nuclear localization of SnoN and v-Ski was not affected by MG132 (Fig. 7C). These findings suggest that MG132-induced cytoplasmic accumulation is unique to c-Ski.
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We next examined whether c-Ski accumulated in the cytoplasm by MG132 affects nuclear translocation of Smad proteins after ligand stimulation (Fig. 7D). C2C12 cells transfected with c-Ski were treated with MG132 for 3 h, and then stimulated with BMP-4 for 1 h. Signals of phospho-Smad1/5/8 were observed in the cytoplasm in c-Ski-transfected cells (arrowhead) whereas they were observed in the nucleus in nontransfected cells (arrow). We thus observed the sequestration of R-Smads by c-Ski wt that is mislocalized in the cytoplasm by MG132.
Mapping of regions in c-Ski required for MG132-induced cytoplasmic accumulation
To elucidate the mechanisms by which nuclear localization of c-Ski is regulated, we tested various c-Ski mutants for MG132-induced cytoplasmic accumulation. c-Ski S2/3 and W274E accumulated in the cytoplasm upon MG132 treatment (data not shown), suggesting that the Smad-binding properties of c-Ski do not affect MG132-induced cytoplasmic accumulation. We also tested various N- or C-terminally truncated mutants of c-Ski (Fig. 8). An N-terminal fragment of c-Ski spanning amino acid residues 1–548 exhibited cytoplasmic accumulation upon MG132 treatment, whereas a c-Ski mutant, spanning residues 1–490 did not. Similarly, c-Ski mutants spanning amino acid residues 94–728 exhibited cytoplasmic accumulation upon MG132 treatment, whereas a c-Ski mutant spanning amino acid residues 211–728 did not. These findings suggested that two regions in c-Ski, amino acid residues 94–210 and 491–548, are required for the cytoplasmic accumulation induced by MG132 treatment.
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Discussion |
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We first identified NLS in c-Ski as a PRKRKLT motif spanning residues 452–458 in human c-Ski. This sequence motif is not conserved in SnoN. Recently, Krakowski et al. (2005) identified NLS in SnoN as K31–K32. Although c-Ski and SnoN exhibit sequence similarity, SnoN has 69 extra amino acid residues in the N-terminus, where the NLS in SnoN is located. Nuclear localization of c-Ski and SnoN thus appears to be determined through different regions of these proteins.
We next constructed a c-Ski mutant (NLS mut) that exhibits cytoplasmic localization and examined the characteristics of cytoplasmic c-Ski. First, we found that the mutated c-Ski migrated more slowly on SDS-PAGE than wild-type c-Ski (c-Ski wt) did. Phosphorylation of c-Ski has also been reported to cause its slower migration on SDS-PAGE (Marcelain & Hayman 2005), but c-Ski (NLS mut) migrated slowly even after phosphatase treatment (the present authors’ unpublished observation). The slower migration of c-Ski (NLS mut) may be the result of structural alteration or protein modification other than phosphorylation. Second, we found that cytoplasmic c-Ski inhibits signaling of TGF-ß as well as BMP. Although c-Ski (NLS mut) was more stable than c-Ski wt, there was little difference in inhibitory effects on TGF-ß/BMP signaling between them when they were expressed at equivalent levels. Using double mutants combining mutations in the Smad binding region and NLS, we found that cytoplasmic c-Ski inhibits Smad signaling through interaction with either R-Smads or Co-Smad.
Prunier et al. (2003) reported that c-Ski interferes with the phosphorylation of Smad2/3 by TGF-ß type I receptor. In our experiments, however, we did not observe inhibition of Smad2 phosphorylation by c-Ski. Instead, c-Ski wt as well as NLS mut caused increase in the level of phospho-Smad2. c-Ski appears to stabilize phospho-Smad2. The reason for the discrepancy between their data and ours is unknown at present. Reed et al. (2001) reported that in metastatic melanomas in which c-Ski is predominantly localized in the cytoplasm, Smad3 failed to translocate to the nucleus in response to TGF-ß. Because Smad3 associates with c-Ski in the cytoplasm, they concluded that c-Ski binding prevents Smad3 translocation. However, they did not present direct evidence that cytoplasmic c-Ski is the factor preventing Smad3 translocation. Kokura et al. (2003) reported that c-Ski inhibits nuclear translocation of Smad2 in the presence of a novel c-Ski binding cytoplasmic protein, C184M. However, it was not determined whether C184M affects Smad2 phosphorylation. In the present study, we clearly demonstrated that cytoplasmic c-Ski sequesters activated Smad complex in the cytoplasm. A similar mechanism has recently been reported for cytoplasmic SnoN (Krakowski et al. 2005).
Although cytoplasmic c-Ski inhibits TGF-ß superfamily signaling through sequestration of activated Smad complex, we found that cytoplasmic c-Ski failed to exhibit some of the activities that c-Ski wt has, including suppression of Smad7 gene expression and activation of myogenin promoter. It appears indispensable for c-Ski to be localized in the nucleus to exhibit these activities. Although the nuclear functions of c-Ski require interaction with Smad4 in some cases (Denissova & Liu 2004), they are unlikely to be related to the Smad signaling that is triggered by type I receptor activation. c-Ski (NLS mut) would thus be useful for segregating Smad-signaling related cellular activities and others including direct regulation of transcription.
Because the subcellular localization of c-Ski affects its biologic activities, it is important to elucidate the mechanisms regulating c-Ski localization. In the present study, we found that the subcellular localization of c-Ski was altered by the treatment of cells with MG132. This effect of MG132 is probably not the result of protection of cytoplasmic c-Ski from degradation, as cytoplasmic c-Ski is more stable than nuclear c-Ski (Fig. 2). MG132 instead appears to affect regulation of c-Ski subcellular localization. One possibility is that MG132 promoted nuclear export of c-Ski. This is, however, unlikely, as leptomycin B did not affect cytoplasmic accumulation. Moreover, MG132 treatment no longer caused cytoplasmic accumulation of c-Ski when new protein synthesis is suppressed by treatment with cycloheximide, suggesting that newly synthesized c-Ski failed to translocate into the nucleus in the presence of MG132. It is thus more likely that a proteasome-sensitive factor suppresses nuclear import of c-Ski, and that in the presence of MG132 this factor is protected from degradation and is therefore active.
Interestingly, we found that two regions of c-Ski (regions C and N) are required for cytoplasmic accumulation in the presence of MG132. Region C spans amino acid residues 491–548. Kokura et al. (2003) reported that cytoplasmic protein C184M sequesters c-Ski in the cytoplasm when C184M is over-expressed in cells. The C184M-interacting region is located at amino acid residues 556–633 of c-Ski, which does not overlap region C. C184M thus may not be one of the proteins regulating c-Ski localization in HeLa cells. Alternatively, it remains possible that some post-translational modification of c-Ski protein, such as phosphorylation, affects its subcellular localization, as reported for Snail (Zhou et al. 2004). In this respect, it is remarkable that region C contains serine-rich sequences (residues 480–536), which are candidate phosphorylation sites in c-Ski (the present authors’ unpublished observation). Region N (spanning residues 94–210), on the other hand, overlaps binding sites for various proteins including nuclear corepressor (Nomura et al. 1999) and FHL-2 (Chen et al. 2003). It remains to be determined how binding proteins or post-translational modifications of c-Ski regulate its subcellular localization.
Upon treatment with MG132, c-Ski accumulates in the cytoplasm, whereas SnoN remains in the nucleus. Of the two regions required for the cytoplasmic accumulation of c-Ski, region N is conserved in SnoN but region C exhibits marked divergence. The difference in localization of c-Ski and SnoN may thus be attributable to region C. These findings suggest that subcellular localization of c-Ski and SnoN is regulated through different mechanisms. Notably, cytoplasmic SnoN has been found in nontumor cells (Krakowski et al. 2005), whereas cytoplasmic c-Ski has been found in tumor cells (Reed et al. 2001; Fukuchi et al. 2004). The differential regulation of nuclear localization of c-Ski and SnoN may be important in endowing them with unique physiologic functions.
At present, the mechanism leading to cytoplasmic localization of c-Ski in some tumor cells remains to be elucidated. Mutations in the NLS of c-Ski may be one possible mechanism. Alternatively, the proteasome-sensitive system regulating localization of c-Ski may not be functioning in those cells. As shown in the present study, cytoplasmic c-Ski is more stable than nuclear c-Ski and still inhibits TGF-ß-induced signal transduction. In addition, it fails to suppress basal expression of Smad7, leading the cells to be more resistant to TGF-ß signaling. Cytoplasmic c-Ski may thus promote oncogenic process more efficiently than c-Ski wt.
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Experimental procedures |
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293T, COS7, HeLa, HepG2, C2C12, and NMuMG cells were obtained from American Type Culture Collection. 293T, COS7, HeLa, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Penicillin 1000 U/mL–1000 µg/mL Streptomycin, Gibco). For C2C12 cells, 20% FBS was used. NMuMG cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS, antibiotics, and 10 µg/mL insulin (Sigma). 293FT cells were purchased from Invitrogen and maintained according to a standard protocol.
DNA constructs and chemicals
Expression constructs encoding ALK5TD-HA, ALK6QD-HA FLAG-Smad2, FLAG-Smad5, FLAG-Smad7, FLAG-c-Ski, and FLAG-SnoN were previously described (Akiyoshi et al. 1999). c-Ski mutants were previously described (Takeda et al. 2004), or generated using a polymerase chain reaction (PCR)-based method. MG132 (Peptide Institute), lactacystin (Sigma), leptomycin B (Calbiochem), cycloheximide (Sigma), TGF-ß1 (R & D Systems), and BMP-4 (R&D Systems) were purchased.
Immunofluorescence labeling
Cells were seeded on eight-well culture slides (BD Falcon) coated with 0.1% gelatin (Cell & Molecular Technologies, Inc.), and were transfected with expression constructs as indicated. At 24 h after transfection, the cells were fixed with methanol/acetone (1 : 1) followed by blocking with Blocking One (Nacalai Tesque). The primary antibodies used were anti-FLAG (M2, x300), anti-c-Ski (see succeeding discussions, x300), and anti-phospho-Smad1/5/8 (Cell Signaling, x200). Anti-c-Ski antibody was prepared as follows: a 19-mer peptide, WPRARPEAAGSEGAAELEP that corresponds to the C-terminus of c-Ski was synthesized and conjugated to keyhole limpet hemocyanin using glutaraldehyde. The conjugate was injected into rabbits in Freund's complete adjuvant, and antiserum was collected from which anti-c-Ski antibody was affinity purified. Alexa 488 or 594-conjugated anti-mouse or rabbit immunoglobulin antibody (Molecular Probes) was used as a secondary antibody. For staining of cell nuclei, propidium iodide (PI) or TOTO-3 (Molecular Probes) was used. All cells were examined under a confocal fluorescence microscope (LSM500, Zeiss).
DNA transfection, immunoprecipitation, and immunoblotting
Cells were transfected using FuGENE6 transfection reagent (Roche Diagnostics) according to the manufacturer's recommendations. Cell lysates were prepared in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1.5% Trasylol, and 1 mM phenylmethylsulfonyl fluoride. After clearing with centrifugation, supernatants were subjected to immunoprecipitation or immunoblotting as described (Komuro et al. 2004). For immunoblotting, anti-FLAG (M2, Sigma), anti-HA (3F10, Roche Diagnostics), anti-tubulin (T9026, Sigma), anti-phospho-Smad2 (Cell Signaling), and anti-phospho-Smad1/5/8 (Cell Signaling) antibodies were used as primary antibodies.
Expression of Venus-fusion proteins in cells
pCS2-Venus (Nagai et al. 2002) was manipulated to delete a stop codon downstream of Venus, which allows to fuse peptide sequence to the C-terminus of the Venus protein. Cassettes of two annealed oligonucleotides encoding putative NLS or its mutant version were inserted between the EcoRI site and the XhoI site. The oligonucleotide sequences used were as follows: PRKRKLT, 5'-AAT TCC AGC CTC GGA AGC GGA AGC TGA CTT AGC-3' and 5'-TCG AGC TAA GTC AGC TTC CGC TTC CGA GGC TGG-3': PNQNQLT, 5'-AAT TCC AGC CTC AGA ACC AGA ACC TGA CTT AGC-3' and 5'-TCG AGC TAA GTC AGG TTC TGG TTC TGA GGC TGG-3'. The expression plasmids were transfected into HeLa cells. Fluorescence images were recorded 24 h after transfection with a fluorescence microscope (IX70–22FL/PH, Olympus).
Pulse-chase analysis
Pulse-chase analysis was performed as previously described (Komuro et al. 2004).
Luciferase assay
Luciferase activity in cell lysates was determined by a dual luciferase reporter assay system (Promega) using a luminometer (AutoLumat LB953, EG & G Berthold) and normalized to seapansy luciferase activity of co-transfected pRL-TK, phRL-TK, or pRL-CMV (Promega). (CAGA)9-MLP-Luc, BRE-Luc, p3TP-lux, and MYO184-Luc were previously described (Edmondson et al. 1992; Carcamo et al. 1994; Dennler et al. 1998; Korchynskyi & ten Dijke 2002). Smad7-Luc was constructed by inserting genomic DNA corresponding to the mouse Smad7 promoter region (–557 ± 112) that was excised with SacI and XhoI (Brodin et al. 2000) and cloned into pGL3-Basic vector (Promega).
Lentiviral production and infection
cDNAs encoding c-Ski and c-Ski (NLS mut) were inserted into the multicloning site (MCS) of the lentiviral vector construct pCSII-EF-MCS-IRES2-Venus (Shibuya et al. 2003) using pENTR according to a standard protocol (Invitrogen). For production of lentiviral vectors, 293FT cells (6 x 106 cells) were transfected using Lipofectamine 2000 (Invitrogen) with pCSII-EF-MCS-IRES2-Venus containing c-Ski or c-Ski (NLS mut) cDNA, pCAG-HIVgp (packaging construct), and pCMV-VSV-G-RSV-Rev (VSV-G- and Rev-expressing construct). The culture supernatants were collected 48 h after transfection, and viral particles were concentrated by centrifugation and used for transduction of NMuMG cells. A lentiviral vector containing the GFP gene was used as a control in some of the experiments.
Growth inhibition assay
Cells were seeded in 24-well plates at a density of 5 x 103 per well and cultured for 24 h. Serum concentration in the medium was then decreased to 1%, and the cells were incubated for another 24 h in the presence or absence of TGF-ß1 (1 ng/mL). The cells were labeled with [3H]thymidine for 30 min. Thymidine incorporation in the TCA-insoluble fraction was analyzed as described (Ebisawa et al. 1999).
Real-time PCR
Total RNA was extracted using RNA-easy (Qiagen) and cDNA was synthesized by Thermoscript real-time (RT)-PCR systems (Invitrogen) following the manufacturer's recommendations. Quantitative RT-PCR analysis was performed using SYBR Green PCR master mix (Applied Biosystems) and ABI PRISM 7000 sequence detection system (Applied Biosystems). The primer sequences used were as follows: mouse Smad7: forward, 5'-ACC GGC TGT TGA AGA TGA CCT, reverse, 5'-TTG CCT CGG ACA GCT CAA TT, mouse Snail, forward, 5'-TTC CTG CTT GGC TCT CTT GGT, reverse, 5'-TAT GGC TCG AAG CAG CTG TGT, mouse GAPDH, forward, 5'-TGC AGT GGC AAA GTG GAG ATT, reverse, 5'-TGC CGT TGA ATT TGC CGT, human c-Ski, forward, 5'-TGT CTG CCG CAG ATT CTC AAC T, reverse, 5'-GGA TGC CCA TGA CTT TGA GGA. Values were normalized to that for GAPDH.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: keiji-miyazawa{at}umin.ac.jp
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References |
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Received: 26 May 2006
Accepted: 10 August 2006
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(B) Immunoblotting detection of c-Ski wt and c-Ski (NLS mut) over-expressed in 293T cells.
(C) Subcellular localization of c-Ski wt and c-Ski (NLS mut) in transfected HeLa cells. Arrowheads indicate localization in nucleus (red), nucleus + cytoplasm (yellow), or cytoplasm (green).
(D) Results in (C) were scored as nucleus (N, red), nucleus + cytoplasm (N + C, yellow), or cytoplasm (C, green). c-Ski-positive cells in three independent fields were counted (n = 171–303), with results reported as mean values.
(E) Subcellular localization of Venus protein fused with the putative NLS sequence or its mutant in HeLa cells. Mutated residues are highlighted in red. Venus proteins were visualized as fluorescence images.
