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1 Department of Immunology, and 2 Department of Dermatology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
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Introduction |
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In the receptor systems of the IL-6-family cytokines, which include IL-6, LIF, CNTF, oncostatin M, and cardiotrophin 1, gp130 is a common receptor subunit responsible for generating multiple signalling pathways. The YXXQ-motif of gp130 is critical not only for the recruitment of STAT3 to the receptor complex and the subsequent tyrosine phosphorylation of STAT3 by activated Jak kinases (Stahl et al. 1995), but also for the activation of an H7-sensitive kinase pathway leading to STAT3 Ser727 phosphorylation (Abe et al. 2001). The latter activity is especially important when cells are stimulated with low concentrations of IL-6 that result in very little activation of ERK1,2 through the YSTV motif (Abe et al. 2001). STAT3 Ser727 phosphorylation enhances the transcription activity of STAT3 by several-fold (Abe et al. 2001; Wen et al. 1995).
The YSTV-motif of gp130 is a docking site for SHP-2 that is responsible for the activation of a Ras/Raf/MEK/ERK-mediated pathway (Fukada et al. 1996). This site is also a docking site for SOCS3, which is induced by a variety of signals, especially STAT3 and ERK1,2-mediated signal, and inhibits the activation of STAT3 (Schmitz et al. 2000; Terstegen et al. 2000). Many of the target genes regulated by the multiple signalling pathways of gp130 have been documented. Among them, some are activated mostly through the STAT3-mediated pathway, which includes the junB, stat3, c-myc, p19INK4D, and pim-1 genes (Fujitani et al. 1994; Ichiba et al. 1998; Kiuchi et al. 1999; Nakajima et al. 1993; Narimatsu et al. 1997; Shirogane et al. 1999). Others are activated mostly through the YSTV-mediated ERK signalling pathway, including the egr-1 gene (Yamanaka et al. 1996). The c-fos gene is activated and c-Fos is accumulated in response to gp130-signals in certain cells (Jenab & Morris 1998). The c-fos gene, however, is different in that both STAT3 and the ERK-mediated pathway co-operatively activate its transcription. Kunisada et al. (1998) showed that dominant-negative STAT3 or a MEK inhibitor, PD98059, inhibit LIF-induced c-fos mRNA expression in a cardiac myocyte cell line. Recently, Yang et al. (2003) showed that STAT3 that was activated by a low concentration of IL-6 binds to the SIE region of the c-fos gene promoter in HepG2 and co-operatively activates the gene with the TPA-induced ERK1,2 pathway.
c-Fos is a member of the Fos family, which includes c-Fos, Fra-1, Fra-2, and FosB; JunB is a member of the Jun family, which includes c-Jun, JunB, and JunD. A Fos family member dimerizes with a member of the Jun family to make an AP-1 transcription factor that binds to the TPA-responsive sequences or the AP-1-binding sites of DNA (Karin 1995). The expression level of c-Fos is regulated at both the transcription level through multiple signalling pathways and the post-translational level (Karin 1995). Although c-Fos is a nuclear protein that has a short half-life in the absence of stimulation, phosphorylation at serines 362 and 374 in the carboxyl terminus stabilizes and extends the half-life of c-Fos to about 2 h (Chen et al. 1996). p90RSK and ERK are responsible for the phosphorylation of c-Fos at Ser362 and Ser374, respectively (Chen et al. 1993). Recently MEK5 and downstream Erk5 were shown to stabilize c-Fos by phosphorylating it at multiple sites that partially overlap with sites phosphorylated by Erk and RSK kinases and that induce AP-1 activity (Terasawa et al. 2003). c-Fos transcription activity is also enhanced by ERK-mediated multiple phosphorylation of the carboxyl-terminal region (Monje et al. 2003; Murphy et al. 2002). The localization of c-Fos is also a target of regulation (Roux et al. 1990). When exponentially growing cells that have been stimulated with serum are deprived of serum for some time, c-Fos leaves the nucleus and gradually disappears (Vriz et al. 1992). Roux et al. (1990) showed that the cytoplasmic localization is dependent on a putative labile inhibitor and that extracellular signals are required for the nuclear translocation of c-Fos. However, the mechanisms by which the localization of c-Fos is determined are not known.
In the present study, we further studied the mechanisms of the co-operation between the STAT3 activating signal and the ERK-mediated signal in activating AP-1, especially c-Fos, in an NIH3T3 cell line expressing a chimeric receptor of G-CSFR fused to the gp130 transmembrane and truncated cytoplasmic domains containing one YXXQ motif, NIH3T3-G108 YRHQ (Abe et al. 2001). We show here that the signal derived from a YXXQ motif in gp130 caused the induction of c-Fos and JunB expression by the STAT3 signal without an apparent increase in AP-1 activity in NIH3T3 cells. Consistent with the low AP-1 activity, c-Fos was predominantly localized to the cytoplasm of NIH3T3 cells stimulated with the YXXQ-signal. We also show that the YXXQ-signal is required when c-Fos is produced for the cytoplasmic retention of the newly formed c-Fos to occur, and a MEK/Erk-signal is required for the nuclear translocation of the c-Fos in the presence of the YXXQ-signal. Thus, we present a new feature of the cooperation between the two main signalling pathways, the YXXQ-derived STAT3 activating signal and a MEK/ERK signal, that induce c-Fos activation.
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Results |
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To investigate the role of the YXXQ-signal in the activation of AP-1, we tested the NIH3T3-G108 YRHQ cell line for the induction of mRNA expression of the c-fos and junB genes, their protein products, and AP-1 activity, in response to G-CSF (granulocyte-colony stimulating factor) or TPA, or to a combination of G-CSF and TPA. We also tested the NIH3T3-G108FRHQ cell line as a control. The chimeric receptor consisted of the extracellular domain of G-CSFR and the transmembrane and proximal cytoplasmic domains of gp130 up to the 108th amino acid residue with the YRHQ motif (G108 YRHQ) or a mutated YRHQ motif, FRHQ (G108FRHQ). The NIH3T3-G108 YRHQ cell line shows STAT3 activation but not ERK1,2 activation in response to G-CSF (Abe et al. 2001). The receptor stimulation caused the transcription and translation of the c-fos and junB genes at levels comparable to those obtained with TPA stimulation (Fig. 1A,B).
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Next, we examined whether the YXXQ-signal causes AP-1 activity using an electrophoretic mobility shift assay (EMSA) with 32P-labelled oligonucleotides containing the collagenase TPA response element (TRE) (Fig. 1C) as well as a reporter assay with a luciferase reporter containing multimerized AP-1-binding sites upstream of the junB gene minimal promoter (Fig. 1D). The receptor stimulation with G-CSF did not significantly increase the amount of AP-1-binding activity detected by the EMSA (Fig. 1C, lanes 2) or the reporter assay (Fig. 1D), but TPA alone increased the AP-1 activity, and simultaneous stimulation with G-CSF and TPA caused strong AP-1 activity in the two assays (Fig. 1C, lane 4 and 1D). The AP-1 activity obtained with the combination of G-CSF and TPA is constantly larger than that with serum stimulation (Fig. 1D). The TRE-binding activity is specific for AP-1, because only the unlabelled oligonucleotides containing TRE, but not those with an irrelevant STAT3-binding site sequence, inhibited the binding of proteins (Fig. 1C, lanes 5-7). The AP-1-binding complexes obtained from the nuclear extracts of cells stimulated with G-CSF and TPA contained c-Fos, JunB, and c-Jun, as revealed by the shifted bands observed when an anti-c-Fos, anti-JunB, or anti-c-Jun antibody was added to the assay (Fig. 1C, lanes 9-11). Taken together, these data indicate that the YXXQ-signal is sufficient for the expression of AP-1 constituent proteins but does not cause AP-1 activation.
c-Fos induced by the YXXQ-signal was predominantly located in the cytoplasm
To explore the reason the YXXQ-signal did not activate AP-1 despite the efficient induction of c-Fos and JunB expression, we examined the subcellular localization of the c-Fos (Fig. 2A) and JunB (Fig. 2B) proteins by immunofluorescence. JunB was predominantly present in the nucleus when its expression was induced by any stimulus (Fig. 2B). In contrast, c-Fos induced by receptor stimulation with G-CSF was predominantly localized to the cytoplasm, but c-Fos induced by TPA stimulation or by TPA and G-CSF was predominantly localized to the nucleus (Fig. 2A). The cytoplasmic localization of the c-Fos induced by the YXXQ-signal was observed at all time points examined from 45 to 300 min, which covers almost the entire time course of the protein expression (Fig. 3). These results suggest that the failure of the YXXQ-signal to activate AP-1 is mainly due to the cytoplasmic localization of the c-Fos protein.
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As shown above, TPA or TPA and G-CSF induced the nuclear accumulation of the c-Fos protein, indicating that a TPA-stimulated signal is likely to alter the subcellular distribution of c-Fos. Therefore, we tested whether TPA stimulation could enable the movement of the cytoplasmic c-Fos (whose expression had been induced by the YXXQ-signal) to enter the nucleus. As shown in Fig. 4B, TPA stimulation for 10 min caused the YXXQ-signal-induced c-Fos to move to the nucleus. This nuclear translocation was efficiently inhibited by pretreating the cells with 30 µM PD98059 (Fig. 4B), a MEK inhibitor. This indicates that a MEK/ERK signal causes the nuclear translocation of cytoplasmic c-Fos. As shown in Fig. 4C, the short-term treatment with TPA did not affect the amount of c-Fos protein, but did affect its migration pattern, most likely by phosphorylating c-Fos in the cytoplasm (Fig. 4C, lanes 3 and 5). This migrational change was inhibited by the pretreatment with PD98059 (Fig. 4C, lane 4). At present, it is not known whether the MEK/ERK-modification of the c-Fos in the cytoplasm is responsible for the translocation of c-Fos.
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To address whether c-Fos and JunB induction by the YXXQ-signal in this cell line was dependent on STAT3 activation, we used the siRNA (small interfering RNA)-mediated gene knock-down method to deplete endogenous STAT3. NIH3T3-G108 YRHQ cells were transfected with either the pU6-mouseSTAT3-siRNA vector or pU6i empty vector together with a pLAT-EGFP vector that localizes to the lipid raft microdomain and allows us to distinguish transfected cells easily. As shown in Fig. 5A, after the cells were stimulated with G-CSF for 15 min, the endogenous mouse STAT3 accumulated in the nucleus, and staining for STAT3 was dramatically reduced in cells transfected with pU6-mouseSTAT3-siRNA but not in cells transfected with pU6i vector (data not shown), demonstrating the efficacy of the STAT3 knock-down by this method. To detect c-Fos clearly, siRNA-transfected cells were stimulated with G-CSF for 80 min, followed by stimulation with TPA for 10 min. As shown in Fig. 5B, the pU6-mouseSTAT3-siRNA-transfected cells did not show c-Fos expression in the nucleus. The control pU6i vector did not affect the YXXQ-signal induced-c-Fos expression (data not shown). The induction of JunB by the YXXQ-signal was also inhibited by the STAT3 siRNA (data not shown), indicating that the c-Fos induction as well as that of JunB by the YXXQ-signal depends on STAT3 activity.
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Chen et al. (1996) and Murphy et al. (2002) have reported that exogenously expressed c-Fos is localized to the nucleus and not the cytoplasm, even when various c-Fos mutants, including c-Fos S362A, c-Fos S374A, and c-Fos T325A/T331A, were expressed in serum-starved cells. Here we examined whether the YXXQ-signal might cause the cytoplasmic translocation of nuclear c-Fos expressed from a constitutive promoter. We transiently transfected NIH3T3-G108 YRHQ cells with the pEF-FLAG-c-Fos expression vector together with the pLAT-EGFP vector. The transfected cells were serum-starved for 24 h and stimulated with G-CSF or left unstimulated for 45 min. Exogenously expressed c-Fos was detected with an anti-FLAG antibody. Figure 6A shows that the FLAG-c-Fos was exclusively localized to the nucleus without stimulation and did not move to the cytoplasm after stimulation (Fig. 6A), indicating that the YXXQ-signal does not cause the cytoplasmic translocation of c-Fos.
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Discussion |
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The subcellular distribution of c-Fos under various conditions has been analysed. In exponentially growing asynchronous cells, c-Fos is localized to the nucleus. It has also been shown to leave the nucleus after serum-starvation for some time and then to gradually disappear (Vriz et al. 1992). The required time after starvation for cytoplasmic translocation appears to be different from cell line to cell line. Roux et al. (1990) showed that this cytoplamic localization of c-Fos may be dependent on a putative labile protein inhibitor in the cytoplasm, since a 1-h treatment with a protein synthesis inhibitor caused nuclear localization of newly formed c-Fos, and because some extracellular signals, including reagents causing cAMP-dependent kinase activation, caused the nuclear translocation of cytoplasmic c-Fos.
In our hands, the 24-h serum-depletion of NIH3T3-G108 YRHQ cells was enough to deplete c-Fos protein to a level that was undetectable by immunostaining in both the cytoplasm and nucleus, as shown in Figs 2 and 3. It is likely that multiple mechanisms exist for the cytoplasmic retention of c-Fos. As shown in Fig. 6A,B, the c-Fos protein, expressed either from a constitutive promoter or an inducible promoter under serum-starvation conditions, was exclusively localized to the nucleus. If the putative cytoplasmic retention mechanism is present in the serum-starved cells, the amounts of c-Fos protein expressed there is likely to far exceed the capacity of the putative mechanism. However, under such a condition, the YXXQ-signal caused the cytoplasmic localization of substantial amounts of c-Fos only when the YXXQ-signal was activated at the time of c-Fos induction. Therefore, this result indicates that the YXXQ-signal activates a cytoplasmic retention mechanism for c-Fos. We can speculate that the YXXQ-signal may allow some inhibitory protein to interact with the newly formed c-Fos molecule in the cytoplasm and that such an inhibitory molecule may itself be a target of the YXXQ-signal. However, this inhibitory mechanism should not be effective for c-Fos already translocated to the nucleus. Interestingly, we showed that this cytoplasmic c-Fos could be translocated into the nucleus by short-term treatment with TPA in a MEK/ERK-dependent manner. As shown in Fig. 4C, this short-term stimulation also caused a mobility shift of c-Fos, most likely due to ERK-dependent phosphorylation. However, this change in the phosphorylation of c-Fos may not be responsible for the release of c-Fos from the cytoplasmic retention mechanism, because various c-Fos phosphorylation mutants expressed from constitutive promoters have been shown to be present in the nucleus in NIH3T3 cells under serum-starvation conditions (Chen et al. 1996; Murphy et al. 2002). Instead, a MEK/ERK signal may disrupt the retention mechanism.
Recently, the phosphorylation of c-Fos by ERK1 (Murphy et al. 2002) or MEK5-ERK5 (Terasawa et al. 2003) at the carboxyl terminal region was shown to enhance the transcriptional activity of c-Fos and AP-1. The induction of c-Fos and JunB cooperatively by the YXXQ-derived STAT3 activating signal and the MEK/ERK-signal both at the transcriptional and post-translational levels is likely to be important for the activation of AP-1. Clearly, further study will be needed to elucidate the intriguing regulation mechanisms for the cytoplasmic retention of c-Fos, the nature of the YXXQ-signal required for this process, and the precise mechanisms that coordinate the cooperation of these two major signalling pathways at both transcriptional and at post-translational levels. The strength and duration of each signal in certain cells stimulated with different concentrations of cytokines would be an important issue in understanding the cooperation between signals.
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Experimental procedures |
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The sources for some of the materials used were as follows: TPA (12-O-tetradecanoyl phorbol-13-acetate) was from Sigma Aldrich. Recombinant human G-CSF was a gift from Kirin Brewery Co. The anti-STAT3 (sc-7179), anti-JunB (sc-46), and anti-c-Fos (sc-52) antibodies were from Santa Cruz Biotechnology. The anti-flag M2 antibody (F3165) was from Sigma Aldrich.
Plasmids
The p6xAP1-Luc contains six repeats of the AP-1 binding sites (5'-TCGACGATCATGACTAAGTTCTATCATGACTAAGTTCTATCATGACTAAGTTTGGTAC-3') x 2 inserted upstream of the minimal junB promoter-Luciferase gene. The wild-type human c-fos cDNA was obtained from the Japan Cancer Research Resources Bank (pSPT-fos cDNA, YG-CO054). The c-fos open reading frame was amplified by PCR from pSPT-fos. For the constitutive expression vector, FLAG-tagged c-fos cDNA was subcloned into pEF-BOS (a gift from Dr S. Nagata) to make pEF-FLAG-human c-Fos. For a tetracycline-inducible vector, the FLAG-tagged c-fos cDNA was subcloned into a plasmid pSP7xTetRE, which contains 7 tandem repeats of the TetR-response element inserted upstream of the minimal junB promoter. All constructs were verified by DNA sequencing. The pLAT-EGFP was made by replacing the DNA fragment coding for the FLAG-SHP-1 of pMIKT-LAT/SHP-1 (a gift from Dr A. Kosugi) with the DNA fragment coding for EGFP taken from pEGFP-1 (Clontech). This pLAT-EGFP codes for a LAT-EGFP fusion protein that is localized to the lipid raft microdomain. The plasmid pU6i-cassette (Genofunction) was previously described (Miyagishi & Taira 2002). The target sequence of murine stat3 mRNA for siRNA is 5'-GAGUCACAUGCCACGUUGG-3'.
Cell culture and transfections
An NIH3T3 cell line expressing truncated G-CSFR-gp130 chimeric receptors with 108 cytoplasmic amino acid residues and a YRHQ motif, NIH3T3-G108 YRHQ, was previously described (Abe et al. 2001). The NIH3T3-G108 YRHQ-Tet-off cell line was made by introducing a tetR-expressing vector, pUHD15-1 (Gossen & Bujard 1992) with pMC1-Neo-polyA (Stratagene) into NIH3T3-G108 YRHQ cells. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL) supplemented with 10% heat-inactivated foetal calf serum (FCS, Sigma Aldrich, St. Louis, MO, USA) and antibiotics. Cells were transiently transfected with different combinations of plasmids using the calcium phosphate precipitation method. Doxycycline was used at 1 µg/ml to suppress the expression of FLAG-c-Fos.
Reporter assay
The transiently transfected NIH3T3-G108 YRHQ cells were stimulated with 10 ng/ml G-CSF, 100 nM TPA, a combination of both G-CSF and TPA, or FCS at 20% or left unstimulated, for 5–6 h. Cell lysates were harvested and assayed for luciferase activity and ß-galactosidase activity as previously described (Kojima et al. 1996).
Immunofluorescence microscopy
NIH3T3-G108 YRHQ cells were plated on coverslips in 6-well plates and transfected with pLAT-EGFP and either pU6-mSTAT3-siRNA or pU6i-cassette control vector. Sixteen hours later, the transfected cells were washed with PBS, re-fed with DMEM containing 10% FCS for 20 h, then serum starved for 36 h with DMEM containing 0.5% FCS. They were then stimulated with 10 ng/ml G-CSF, 100 nM TPA, or a combination of both G-CSF and TPA, or left unstimulated, for the indicated time. After being washed with PBS, the cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.5% Triton-X, and blocked with 3% skim milk. They were then incubated sequentially with the appropriate primary antibody for 1–2 h and the appropriate secondary antibody for 1 h. The primary antibodies to c-Fos, JunB, and STAT3 were used at a 1 : 200 dilution and anti-FLAG monoclonal antibody, M2, was used at 5 µg/ml in 3% skim milk. After incubation with the antibodies, the cells were incubated in 0.2 µg/ml DAPI for 5 min, washed with PBS containing 0.1% Tween20 (TPBS), and rapidly rinsed in water before being mounted in PermaFluor aqueous mounting medium. The coverslips were analysed by a confocal laser-scanning microscope (Carl Zeiss).
Western blotting
After stimulation, the cells were lysed with ice-cold lysis buffer (50 mM HEPES pH 7.5, 1% NP-40, 300 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1 mM sodium vanadate, 0.1 mM PMSF). The cell lysates were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with either an anti-c-Fos or anti-JunB antibody. Signals were detected with the ECL system (Amersham Biosciences).
Electrophoretic mobility shift assays (EMSA)
Electrophoretic mobility shift assays were performed as previously described (Kojima et al. 1996). Briefly, Oligonucleotides were labelled by filling in 5' extensions with a Klenow enzyme using [
-32P]dCTP. Nuclear extracts (10 µg) were incubated in a final volume of 20 µl (10 mM HEPES, pH 7.9, 80 mM NaCl, 10% Glycerol, 1 mM dithiothreitol, 1 mM EDTA, 100 µg/ml poly(dI-dC)poly(dI-dC)) with a probe (10000 c.p.m., 0.5–1 ng) for 30 min at room temperature. In the competition analysis, nuclear extracts were incubated with a 20- or 100-fold molar excess of cold oligonucleotides, or incubated with 1 µg of an antibody for an hour on ice before the mixing with 32P-labelled probe. The protein-DNA complexes were resolved on a 4.5% non-denaturing polyacrylamide gel containing 2.5% glycerol in 0.25 x TBE (1xTBE is 0.13 M Tris base, 0.12 M boric acid, and 2.0 mM EDTA, pH 8.8) at room temperature. The gels were dried and subjected to Fuji BAS 2500 Bio-image Analyser (Fuji Film, Tokyo, Japan). Oligonucleotides used for the probe or competition assay were as follows: Collagenase TRE (A) 5'-AGCTTGATGAGTCAGCCG-3' and (B) 3'-ACTACTCAGTCGGCCTAG-5'; rat alpha2-macroglobulin APRE (acute phase response element) (A) 5'-GCGCCTTCTGGGAATTCCTA-3' and (B) 3'-GAAGACCCTTAAGGATCGCG-5'.
Northern blotting
Northern blotting was performed as previously described (Kiuchi et al. 1999). The probes used were the human c-fos cDNA from the Japan Cancer Research Resources Bank (2.1 kb, the EcoRI fragment containing an ORF and both 5'- and 3' UTR), human junB (2.1 kb, the EcoRI fragment), and CHOB (0.6 kb, the EcoRI-BamHI fragment).
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Acknowledgements |
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Footnotes |
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These two authors contributed equally. 
* Correspondence: E-mail: knakajima{at}med.osaka-cu.ac.jp |
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Received: 4 November 2003
Accepted: 15 December 2003
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  T. Nishikawa, K. Hagihara, S. Serada, T. Isobe, A. Matsumura, J. Song, T. Tanaka, I. Kawase, T. Naka, and K. Yoshizaki Transcriptional Complex Formation of c-Fos, STAT3, and Hepatocyte NF-1{alpha} Is Essential for Cytokine-Driven C-Reactive Protein Gene Expression J. Immunol., March 1, 2008; 180(5): 3492 - 3501. [Abstract] [Full Text] [PDF]
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 L. Al-Khalili, K. Bouzakri, S. Glund, F. Lonnqvist, H. A. Koistinen, and A. Krook Signaling Specificity of Interleukin-6 Action on Glucose and Lipid Metabolism in Skeletal Muscle Mol. Endocrinol., December 1, 2006; 20(12): 3364 - 3375. [Abstract] [Full Text] [PDF]
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 R. R. Nair, J. Solway, and D. D. Boyd Expression Cloning Identifies Transgelin (SM22) as a Novel Repressor of 92-kDa Type IV Collagenase (MMP-9) Expression J. Biol. Chem., September 8, 2006; 281(36): 26424 - 26436. [Abstract] [Full Text] [PDF]
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M. Arnold, A. Nath, D. Wohlwend, and R. H. Kehlenbach Transportin Is a Major Nuclear Import Receptor for c-Fos: A NOVEL MODE OF CARGO INTERACTION J. Biol. Chem., March 3, 2006; 281(9): 5492 - 5499. [Abstract] [Full Text] [PDF]
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T. Tanos, M. J. Marinissen, F. C. Leskow, D. Hochbaum, H. Martinetto, J. S. Gutkind, and O. A. Coso Phosphorylation of c-Fos by Members of the p38 MAPK Family: ROLE IN THE AP-1 RESPONSE TO UV LIGHT J. Biol. Chem., May 13, 2005; 280(19): 18842 - 18852. [Abstract] [Full Text] [PDF]
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 H. Kojima, T. Sasaki, T. Ishitani, S.-i. Iemura, H. Zhao, S. Kaneko, H. Kunimoto, T. Natsume, K. Matsumoto, and K. Nakajima STAT3 regulates Nemo-like kinase by mediating its interaction with IL-6-stimulated TGF{beta}-activated kinase 1 for STAT3 Ser-727 phosphorylation PNAS, March 22, 2005; 102(12): 4524 - 4529. [Abstract] [Full Text] [PDF]
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