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• W2148252804 abstract "Article1 February 2002free access The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription Jukka Westermarck Corresponding Author Jukka Westermarck EMBL, D-69117 Heidelberg, Germany Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520 Turku, Finland Search for more papers by this author Carsten Weiss Carsten Weiss EMBL, D-69117 Heidelberg, Germany Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Rainer Saffrich Rainer Saffrich EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Jürgen Kast Jürgen Kast EMBL, D-69117 Heidelberg, Germany Present address: Biomedical Research Centre, Vancouver, BC, Canada, V6T 1Z3 Search for more papers by this author Anna-Maria Musti Anna-Maria Musti EMBL, D-69117 Heidelberg, Germany Università della Calabria, I-87100 Rende (CS), Italy Search for more papers by this author Matthias Wessely Matthias Wessely EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Wilhelm Ansorge Wilhelm Ansorge EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Bertrand Séraphin Bertrand Séraphin EMBL, D-69117 Heidelberg, Germany CGM-CNRS, F-91198 Gif sur Yvette Cedex, France Search for more papers by this author Matthias Wilm Matthias Wilm EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Benigno C. Valdez Benigno C. Valdez Department of Pharmacology, Baylor College of Medicine, Houston, TX, 77030 USA Search for more papers by this author Dirk Bohmann Corresponding Author Dirk Bohmann EMBL, D-69117 Heidelberg, Germany Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Jukka Westermarck Corresponding Author Jukka Westermarck EMBL, D-69117 Heidelberg, Germany Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520 Turku, Finland Search for more papers by this author Carsten Weiss Carsten Weiss EMBL, D-69117 Heidelberg, Germany Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Rainer Saffrich Rainer Saffrich EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Jürgen Kast Jürgen Kast EMBL, D-69117 Heidelberg, Germany Present address: Biomedical Research Centre, Vancouver, BC, Canada, V6T 1Z3 Search for more papers by this author Anna-Maria Musti Anna-Maria Musti EMBL, D-69117 Heidelberg, Germany Università della Calabria, I-87100 Rende (CS), Italy Search for more papers by this author Matthias Wessely Matthias Wessely EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Wilhelm Ansorge Wilhelm Ansorge EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Bertrand Séraphin Bertrand Séraphin EMBL, D-69117 Heidelberg, Germany CGM-CNRS, F-91198 Gif sur Yvette Cedex, France Search for more papers by this author Matthias Wilm Matthias Wilm EMBL, D-69117 Heidelberg, Germany Search for more papers by this author Benigno C. Valdez Benigno C. Valdez Department of Pharmacology, Baylor College of Medicine, Houston, TX, 77030 USA Search for more papers by this author Dirk Bohmann Corresponding Author Dirk Bohmann EMBL, D-69117 Heidelberg, Germany Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Author Information Jukka Westermarck 1,2, Carsten Weiss1,3, Rainer Saffrich1, Jürgen Kast1,4, Anna-Maria Musti1,5, Matthias Wessely1, Wilhelm Ansorge1, Bertrand Séraphin1,6, Matthias Wilm1, Benigno C. Valdez7 and Dirk Bohmann 1,3 1EMBL, D-69117 Heidelberg, Germany 2Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520 Turku, Finland 3Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA 4Present address: Biomedical Research Centre, Vancouver, BC, Canada, V6T 1Z3 5Università della Calabria, I-87100 Rende (CS), Italy 6CGM-CNRS, F-91198 Gif sur Yvette Cedex, France 7Department of Pharmacology, Baylor College of Medicine, Houston, TX, 77030 USA *Corresponding authors: E-mail: [email protected] E-mail: [email protected] The EMBO Journal (2002)21:451-460https://doi.org/10.1093/emboj/21.3.451 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfered with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells. Introduction The AP-1 family transcription factor c-Jun plays a pivotal role in the genetic responses of cells to a number of extracellular stimuli, including stress insults, apoptotic and differentiation signals. Many of these signals are transduced to c-Jun by signaling pathways that culminate in the activation of Jun-N-terminal kinase (JNK), causing the phosphorylation and activation of c-Jun (Leppä and Bohmann, 1999; Davis, 2000). In spite of a large body of literature on the topic, the molecular mechanism by which the transcriptionally active form of c-Jun stimulates mRNA synthesis is still incompletely understood, but it presumably involves the recruitment of specific co-factors. Several c-Jun-interacting proteins have been described (for a review see Chinenov and Kerppola, 2001). Examples are the co-activators CBP and p300, which bind to the N-terminal transactivation domain of c-Jun in vitro and enhance AP-1-mediated promoter activation (Chinenov and Kerppola, 2001). Remarkably, none of the proteins reported to bind to the transactivation domain of c-Jun and to facilitate c-Jun-mediated transcription has been shown to interact with c-Jun in in vivo binding assays. In that regard, a further development of methods for the identification of proteins that participate in c-Jun-mediated gene activation in vivo is required. Recent progress in mass spectrometric protein sequencing technology as well as the rapid growth of protein and genome databases have made direct approaches to map protein–protein interactions feasible. Because the identification of interacting proteins by a biochemical route is not based on a transcriptional read-out, as is the case in yeast two-hybrid assays, it is also amenable to the study of transcription factor complexes. Results Identification of RHII/Gu RNA helicase as an interaction partner of c-Jun by tandem affinity purification In order to isolate proteins that interact with the transactivation domain of c-Jun in human cells, we employed the tandem affinity purification (TAP) method (Rigaut et al., 1999). Briefly, the protein of interest is fused to two ligand-binding domains, one derived from a calmodulin-binding peptide, the other from protein A (Figure 1A). This construction endows the bait protein with high affinity for both calmodulin and IgG affinity purification resins. The calmodulin-binding peptide and protein A affinity tags are separated by the recognition sequence for tobacco etch virus (TEV) protease, permitting proteolytic elution of the fusion protein from the IgG affinity resin (Rigaut et al., 1999; Puig et al., 2001). The tagged protein, potentially in a complex with interacting factors, can be purified from crude extracts by two consecutive affinity chromatographic steps. Figure 1.Identification of the c-Jun-binding protein complex by TAP purification. (A) Schematic representation of c-Jun (top) and c-JunTAP structures (bottom). The region of the DNA-binding domain (DBD) and the leucine zipper dimerization domain (ZIP) in c-Jun has been replaced with the TAP tag, consisting of a calmodulin-binding peptide (CBP), a consensus TEV cleavage site (TEV) and the IgG-binding domain of protein A (Prot. A). The position of regulatory JNK MAP kinase phosphorylation sites is indicated. (B) Analysis of the c-JunTAP-interacting proteins. Proteins of the TAP eluate from untransfected 293 mock-treated cells or cells transfected with the c-JunTAP expression vector were resolved on an SDS–polyacrylamide gel and silver stained. The protein bands from which NDHII, RHII/Gu and hnRNP M peptide sequences were identified are indicated. In both lanes, a prominent band at ∼50 kDa derived from human IgG leeched off the first column of the TAP purification. In the c-JunTAP lane, a prominent band at ∼30 kDa represents the bait after enzymatic removal of the protein A affinity tag by TEV. (C) Identification of the 84 kDa protein band as RHII/Gu by tandem mass spectrometry. The mass spectrum of the extracted peptides after tryptic digestion is shown in the top panel. Ions from various sources were observed and evaluated based upon the following criteria. Known autoproteolysis products of trypsin (T) and common keratin peptides (K) were identified by comparison of the spectrum with a blank based on their mass-to-charge values and charge states, whereas singly charged ions due to polymer contamination of the solvents used were determined by their characteristic 44 Da repeats (P), all of which were excluded from tandem mass spectrometric analysis. Finally, singly charged peptide ions (S) known to provide limited sequencing information due to poor fragmentation were not considered, while three doubly charged peptide ions marked by arrows were identified and fragmented individually. From all three peptides, tandem mass spectra were acquired that allowed RHII/Gu to be identified independently. An example of a tandem mass spectrum acquired from a doubly charged ion at m/z 834.8 (see enlarged part of the mass spectrum as inset in top panel) is shown in the bottom panel. The peptide sequence was derived from the mass difference between adjacent fragments of the y-ion series. The peptide sequences thus determined and their corresponding location in the RHII/Gu protein sequence are: EGAFSNFPISEETIK (203–217), APQVLVLAPTR (277–287) and IGVPSATEIIK (581–591). (D) Verification of the interaction between c-Jun and interacting proteins by western blot analysis. Nuclear proteins from HT-1080 cells stably transfected with the c-Jun1–223TAP expression construct or from control cells bearing empty expression vector were subjected to TAP purification. Aliquots of nuclear proteins and TEV eluates were analyzed by western blot using specific antibodies against human c-Jun, NDHII, RHII/Gu and hnRNP M. Note that the TAP-tagged c-Jun protein derivatives in the starting material migrate at slightly higher apparent molecular mass than the endogenous c-Jun. Download figure Download PowerPoint We developed a mammalian expression vector that codes for a fusion protein consisting of amino acids 1–223 of c-Jun linked to the TAP tandem affinity purification domain (c-Jun1–223TAP, Figure 1A). For a typical experiment such as the one shown in Figure 1B, 108 293 cells were transiently transfected with the c-Jun1–223TAP expression construct. After 36 h, nuclear extracts were prepared and applied to dual affinity chromatography according to the TAP protocol. The purified eluates contained a number of polypeptide species that co-purified specifically with the TAP-tagged c-Jun bait, but were absent in the fraction purified from control cells (Figure 1B). These products were therefore candidates for c-Jun-interacting proteins. Bands representing putative c-Jun-binding proteins were purified by SDS–PAGE and analyzed by mass spectrometric peptide sequencing. Comparison of the obtained peptide sequences with protein databases identified several proteins that previously had been linked to mRNA metabolism and transcription. Here we report the identification of three proteins, the DNA/RNA helicase NDHII, hnRNP M and the RNA helicase RHII/Gu, as novel interaction partners for c-Jun. The characterization of other interacting proteins will be reported elsewhere. The identification of RHII/Gu (Valdez et al., 1996) was based on sequences obtained from three peptides showing complete identity to the human RHII/Gu protein sequence (see Figure 1C and figure legend). The other interaction partners were identified in a similar manner. To verify the identity of NDHII, RHII/Gu and hnRNP M as c-Jun-binding partners, we performed a small-scale TAP experiment using an HT-1080 human fibrosarcoma line that carries the c-JunTAP expression vector integrated into its genome. Immunoblot analyses with NDHII-, RHII/Gu- and hnRNP M-specific antibodies revealed cross-reactive protein species of the expected molecular masses in the relevant eluate fraction (Figure 1D, upper panel). We conclude that NDHII, RHII/Gu and hnRNP M were retained on the column by means of their interaction with c-JunTAP. These results validate the mass spectrometric identification of NDHII, RHII/Gu and hnRNP M as c-Jun-binding proteins. Furthermore, the experiment confirms that the interaction of c-Jun with the analyzed proteins is not restricted to 293 cells and occurs when the c-JunTAP fusion protein is expressed at levels comparable with endogenous c-Jun as they are present in the stably transfected HT-1080 cells. DEAD-box RNA helicases have been identified previously as auxiliary factors in transcription and translation processes (Eisen and Lucchesi, 1998; Luking et al., 1998; Linder and Daugeron, 2000). NDHII, for example, binds CREB, a leucine zipper transcription factor and a distant relative of c-Jun. NDHII mediates an interaction between RNA polymerase II (pol II) and CREB-binding protein (CBP) and supports CREB-mediated transcription activation in reporter gene assays, an effect that requires the ATPase function of the enzyme (Nakajima et al., 1997). It is thus not surprising that NDHII also interacts with c-Jun. It is conceivable that this enzyme supports c-Jun transcription in a manner comparable with its role in CREB target gene activation. Interestingly, one other newly identified c-Jun interaction partner, RHII/Gu, is a human DEAD-box RNA helicase (Figure 1B and D). Unlike NDHII, however, RHII/Gu has not been implicated previously in RNA pol II-mediated transcription. The presence of RHII/Gu in a complex with c-Jun thus prompted further studies on the role of this enzyme in AP-1-regulated gene expression. RHII/Gu binds c-Jun directly To study whether the interaction between RHII/Gu and c-Jun is mediated by direct protein–protein contact, in vitro pull-down assays were performed. Different bacterially expressed GST–RHII/Gu fusion proteins were immobilized on glutathione–beads and subsequently incubated with various sources of c-Jun. Immunoblot analyses revealed that both bacterially expressed c-Jun (c-JunHis6) and c-Jun present in 293 cell nuclear extracts were retained specifically on the immobilized C-terminal domain of RHII/Gu (amino acids 646–801; Figure 2A, lanes 3 and 6). However, no interaction of c-Jun with the N-terminal part of RHII/Gu or with GST alone could be detected (Figure 2A, lanes 1, 2, 4 and 5). This result shows that the interaction observed by the TAP method can also be reconstituted in vitro and, importantly, that it is direct and does not rely on any intermediary proteins that might have been present in 293 or HT-1080 cells. Furthermore, these results show that RHII/Gu protein may interact with the non-fused wild-type form of c-Jun present in nuclear extracts. Figure 2.Direct protein–protein interaction between RHII/Gu and c-Jun. (A) Mapping of the c-Jun-binding domain of RHII/Gu. Bacterially expressed GST–RHII/Gu proteins were immobilized on glutathione–beads and incubated with either bacterially expressed full-length c-JunHis6 protein or with nuclear proteins from unstimulated 293 cells. After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by western blotting with c-Jun (top) or GST antibodies (bottom). A schematic representation of the structure of RHII/Gu and summary of the binding data are shown below. (B) The interaction between RHII/Gu and c-Jun is not mediated by DNA or RNA. GST pull-down experiments between GST–RHII/Gu 749–801 and c-JunHis6 were performed in the absence or presence of ethidium bromide (10 or 50 μg/ml), 40 μg/ml RNase A or NaCl (300 or 600 mM). Download figure Download PowerPoint Analysis of deletion mutants of RHII/Gu in the pull-down assay revealed that the c-Jun interaction domain on RHII/Gu resides in the most C-terminal part of the protein between amino acids 749 and 801 (Figure 2A). Importantly, the interaction between c-Jun and GST–Gu 749–801 was not affected by addition of high concentrations of ethidium bromide or RNase A, or by 600 mM NaCl (Figure 2B), demonstrating that the interaction between the recombinant proteins is not mediated by contaminating DNA or RNA, and is stable at moderately high ionic strength. Interaction between endogenous c-Jun and RHII/Gu After showing that RHII/Gu and c-Jun interact in vitro or after overexpression of c-Jun in transfected cells, it was important to show that the interaction between the proteins at their endogenous expression levels also occurs in cells. To this end, we used two independent approaches. First, we performed co-immunoprecipitation studies on nuclear extracts from non-transfected 293 and HT-1080 cells. Immunoblotting confirmed that c-Jun antiserum efficiently co-immunoprecipitated endogenous RHII/Gu and c-Jun from both cell lines, whereas no precipitation of either of the proteins was observed with control pre-immune serum (PI) (Figures 3A and 4B). This experiment demonstrates that c-Jun and RHII/Gu proteins interact in human cells at their endogenous level of expression. Figure 3.Interaction between endogenous RHII/Gu and c-Jun proteins in 293 and HT-1080 cells. (A) RHII/Gu specifically co-immunoprecipitates with c-Jun. Nuclear extracts from non-transfected subconfluent 293 cells were subjected to immunoprecipitation (IP) with either specific rabbit anti-c-Jun antiserum or rabbit pre-immune serum (PI). Immunoprecipitated proteins were analyzed by western blotting (IB) using anti-RHII/Gu or anti-c-Jun antibodies. Similar amounts of starting material were used in each lane. (B) RHII/Gu and c-Jun form a protein complex on AP-1-binding sites. Whole-cell extracts from non-transfected subconfluent HT-1080 cells were incubated with 5′-biotinylated double-stranded 25mer oligonucleotides carrying either a consensus or a mutated AP-1-binding site. The oligonucleotides were recovered using streptavidin-conjugated magnetic beads. Bound proteins were visualized by western blot using anti-RHII/Gu or anti-c-Jun antibodies. Similar amounts of starting materials were used in each lane. Both panels show representative examples of two or three experiments with similar results. Download figure Download PowerPoint Figure 4.The interaction between c-Jun and RHII/Gu is increased upon stress signaling. (A) Phosphorylation of c-Jun in JNK phosphorylation sites is not required for interaction between c-Jun and RHII/Gu. Subconfluent 293 cells were transiently co-transfected with vectors directing the expression of ΔMEKK1 and different c-JunTAP constructs, containing either wild-type c-Jun sequences (Wt), or derivatives thereof in which the MAP kinase phosphorylation sites at position 63, 73, 91 and 93 were mutated to alanine (Ala) or aspartic acid (Asp) residues. Nuclear proteins were prepared and subjected to IgG affinity purification and TEV cleavage. Aliquots of nuclear proteins and TEV eluates were analyzed by western blot using specific antibodies against human c-Jun or RHII/Gu. (B) The interaction between endogenous c-Jun and RHII/Gu proteins is increased upon stress signaling. Nuclear extracts from untreated or anisomycin-treated (5 μg/ml, 1 h) subconfluent HT-1080 cells were subjected to immunoprecipitation with either c-Jun antiserum or rabbit pre-immune serum (PI). The amounts of c-Jun and RHII/Gu in the immunoprecipitates were analyzed by western blotting with RHII/Gu and c-Jun antibodies. Phosphorylation of c-Jun in the anisomycin-treated samples is apparent by the mobility shift of the protein. Similar amounts of starting material were used for each lane. (C) Anisomycin-induced increase of RHII/Gu binding is independent of c-Jun phosphorylation state. Subconfluent HT-1080 cells stably expressing c-Jun1–223AspTAP were treated for 1 h with anisomycin (5 μg/ml), or left untreated, and nuclear proteins were subjected to IgG affinity purification and TEV cleavage. The amounts of c-Jun and RHII/Gu proteins in the TEV eluate and nuclear proteins used as a starting material were examined by western blot using RHII/Gu and c-Jun antibodies. Phosphorylation of endogenous c-Jun in the anisomycin-treated samples is discernable by mobility shift of the protein. All panels show representative examples of two or three experiments with similar results. Download figure Download PowerPoint In the next experiment, we used a DNA affinity purification assay to examine whether RHII/Gu interacts with DNA-bound c-Jun. A biotinylated double-stranded DNA oligonucleotide probe, containing a consensus AP-1-binding site, retrieved both c-Jun and RHII/Gu from extracts of non-transfected HT-1080 and 293 cells (Figure 3B and data not shown). In contrast, no significant binding of either c-Jun or RHII/Gu proteins was observed with a control oligonucleotide carrying a mutated AP-1-binding site (Figure 3B). This finding indicates that the binding of c-Jun to DNA and to RHII/Gu is not mutually exclusive, and c-Jun could conceivably deliver RHII/Gu to a transcription unit. Anisomycin stimulates c-Jun–RHII/Gu interaction The part of c-Jun that is present in c-Jun1–223TAP carries regulatory phosphorylation sites for the MAP kinase JNK. To examine whether the interaction between RHII/Gu and c-Jun might be influenced by the phosphorylation state of the latter, the binding of RHII/Gu to different phosphorylation site mutants of c-Jun1–223TAP was examined (Figure 4A). In c-Jun1–223AspTAP, the JNK phosphorylation sites were replaced with aspartic acid to create a mimic of phosphorylated c-Jun (Treier et al., 1995). A c-Jun1–223AlaTAP construct, with alanine substitutions of the phosphorylation sites, was generated in order to create an uninduced, inactive form of c-Jun. 293 cells were transiently transfected with c-Jun1–223AlaTAP, c-Jun1–223TAP or c-Jun1–223AspTAP. In addition, these cells received an expression vector for ΔMEKK1, a constitutively active JNKKK that induces high JNK activity and phosphorylation of both endogenous c-Jun and wild-type c-Jun1–223TAP protein (Figure 4A). Probing of the eluate fractions with anti-RHII/Gu serum showed that all three c-Jun1–223TAP proteins were expressed and recovered at comparable levels and co-purified with approximately the same amounts of RHII/Gu (Figure 4A, lanes 2–4). This finding indicates that c-Jun phosphorylation does not influence binding to RHII/Gu. However, it does not exclude the possibility that the interaction between the proteins might be regulated by signals that increase the transcriptional activity of c-Jun, in a manner that is not mediated by the c-Jun phosphorylation sites. To test this idea, non-transfected HT-1080 cells were treated with anisomycin and the amount of RHII/Gu in c-Jun co-immunoprecipitates was measured by western blotting. Anisomycin treatment induces potent activation of the JNK pathway and prominent phosphorylation of the immunoprecipitated c-Jun protein (Figure 4B). Interestingly, more RHII/Gu was co-immunoprecipitated with c-Jun from anisomycin-treated cells, whereas the amount of c-Jun in the immunoprecipitate was identical in treated and control cells. These results suggest that anisomycin-induced signaling regulates the interaction between c-Jun and RHII/Gu. To confirm that this effect is independent of c-Jun phosphorylation, as suggested by the experiment shown in Figure 4A, we tested whether anisomycin treatment would also increase RHII/Gu binding to a c-Jun derivative that lacks the relevant phosphorylation sites. For this purpose, we used a stable HT-1080 cell line expressing the c-Jun1–223AspTAP fusion protein, and performed TAP purification from untreated or anisomycin-treated cells. As shown in Figure 4C, anisomycin treatment significantly increased binding of endogenous RHII/Gu to c-Jun1–223AspTAP, but did not cause increased expression of RHII/Gu protein or an elevated recovery of c-Jun1–223AspTAP in the eluate of the affinity column (Figure 4C). JNK signaling regulates subnuclear localization of RHII/Gu The results above show that the stoichiometry of interaction between endogenous c-Jun and RHII/Gu proteins can be increased by cellular signaling that induces JNK activity. As this effect does not depend on c-Jun phosphorylation on the known JNK substrate sites, it is possible that it is mediated by a JNK-induced change in the properties of RHII/Gu. RHII/Gu protein has been reported previously to be a predominantly nucleolar protein. Interestingly, treatment of cells with cytotoxic drugs results in translocation of RHII/Gu from nucleolus to nucleoplasm (Perlaky et al., 1997). This suggests that RHII/Gu has both nucleolar and nucleoplasmic functions. Thus, we investigated whether JNK activation might also cause accumulation of RHII/Gu in the nucleoplasm, which would provide an explanation for the JNK-dependent stimulation of c-Jun–RHII/Gu interaction. We found, in addition to the prevalent nucleolar stores, a significant fraction of RHII/Gu in the nucleoplasm of both 293 and HT-1080 cells. Next, we examined if activation of the JNK pathway might cause an enhanced translocation of RHII/Gu into the nucleoplasm. To this end, HT-1080 cells were seeded on glass coverslips and, after treatment with anisomycin (5 μg/ml), UVC (20 J/m2) or actinomycin D (0.2 μM) for 1 h, cells were fixed and immunostained with RHII/Gu antisera. As reported earlier, actinomycin D treatment induced significant translocation of RHII/Gu from nucleolus to nucleoplasm, resulting in uniform nucleoplasmic staining (Figure 5A). Interestingly, treatment of cells with anisomycin and UVC also increased nucleoplasmic levels of RHII/Gu (Figure 5A). Figure 5.JNK activity regulates subnuclear localization of RHII/Gu. (A) Chemical activators of JNK signaling cause nucleoplasmic retention of RHII/Gu in HT-1080 cells. Serum-starved HT-1080 were left untreated (CTL), treated with anisomycin (5 μg/ml), UVC (20 J/m2) or actinomycin D (0.2 μM) for 1 h, cells were fixed, immunostained with RHII/Gu antisera and analyzed by fluorescence microscopy. (B) Overexpression of constitutively active MEKK1 causes nucleoplasmic retention of RHII/Gu in HT-1080 cells. HT-1080 cells were transiently co-transfected with an expression construct coding for ΔMEKK1 together with RHII/Gu–GFP, and after 24 h cells were serum starved for 12 h, after which subcellular localization of RHII/Gu–GFP fusion protein was studied by fluorescence microscopy. Hoechst staining was used to visualize the morphology of nuclei. Shown are representative images of four experiments showing similar results. Download figure Download PowerPoint Even though anisomycin and UVC treatments have been used as prototypical activators of JNK signaling, they influence multiple cellular processes. In order to study specifically the role of JNK signaling in RHII/Gu translocation, HT-1080 cells were transfected with RHII/Gu–green fluorescent protein (GFP) alone or together with an expression vector for ΔMEKK1. Activation of JNK signaling by ΔMEKK1 resulted in markedly increased nucleoplasmic staining of RHII/Gu–GFP (Figure 5B). Taken together, these findings provide evidence that in addition to the well-established function of activating c-Jun by direct phosphorylation, JNK also promotes the translocation of RHII/Gu from the nucleolus to the nucleoplasm. This mechanism provides a plausible explanation for the observed stimulation of the c-Jun and RHII/Gu interaction in response to anisomycin-induced JNK signaling. RHII/Gu contributes to transcription activation by c-Jun The observation that RHII/Gu preferentially interacts with c-Jun in vivo in conditions that coincide with target gene activation raises the possibility that it supports this process. To test this idea, we performed experiments in which HT-1080 cells were microinjected with an expression construct for a c-JunAsp–estrogen receptor fusion protein (c-JunAspER) and an AP-1-responsive LacZ reporter construct. In addition, the injected cells received an anti-RHII/Gu antibody, which blocks helicase activity, or a control antibody. The c-JunAspER was used for these experiments because" @default.
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• W2148252804 title "The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription" @default.
• W2148252804 cites W1575542444 @default.
• W2148252804 cites W1579930914 @default.
• W2148252804 cites W1635552303 @default.
• W2148252804 cites W1979533910 @default.
• W2148252804 cites W2001617515 @default.
• W2148252804 cites W2002223631 @default.
• W2148252804 cites W2002282120 @default.
• W2148252804 cites W2005650892 @default.
• W2148252804 cites W2007320902 @default.
• W2148252804 cites W2009626338 @default.
• W2148252804 cites W2011282682 @default.
• W2148252804 cites W2013467101 @default.
• W2148252804 cites W2022190092 @default.
• W2148252804 cites W2035398832 @default.
• W2148252804 cites W2046764234 @default.
• W2148252804 cites W2048653746 @default.
• W2148252804 cites W2065868028 @default.
• W2148252804 cites W2076485183 @default.
• W2148252804 cites W2078594804 @default.
• W2148252804 cites W2085795760 @default.
• W2148252804 cites W2094646101 @default.
• W2148252804 cites W2119245063 @default.
• W2148252804 cites W2135116219 @default.
• W2148252804 cites W2142242441 @default.
• W2148252804 cites W2149676146 @default.
• W2148252804 cites W2171171901 @default.
• W2148252804 cites W2329404090 @default.
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