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Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt, Germany
Received 27 October 2004/ Returned for modification 9 December 2004/ Accepted 15 December 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Homeodomain proteins (HDPs) have been shown to exert key developmental functions throughout the metazoa since defects in the evolutionary conserved homeobox genes were shown to cause many human disorders and aberrant animal phenotypes (reference 57 and references therein). Homeobox-containing genes encode transcription factors and are characterized by the homeodomain (HD), a motif that directs specific DNA binding to regulate the expression of target genes. Homeobox genes are grouped into several subclasses according to the primary structure of their homeodomain and its flanking sequences (reference 12 and references therein). Among the paired-like subclass, the paired-like CVC (PLC)-HDPs are characterized by a conserved CVC domain and can be grouped into the Vsx-1 and Vsx-2 family (32, 40), containing orthologs from several species. PLC-HDPs appear to play a particular role in ocular development (references 7, 21, 38, and 41 and references therein) and execute their functions by binding to the conserved locus control region (LCR), located upstream of the transcription initiation site of the red opsin gene, and thus specify the development and differentiation of cone photoreceptors and a subset of retinal inner nuclear layer bipolar cells (references 16 and 49 and references therein). The observation that null mutations in Chx10 cause congenital microphthalmia, including small eyes, cataracts, iris coloboma, and blindness in humans (42), mice (5), and zebra fish (2), underscores the importance of the PLC-HDP gene family for retinogenesis. Thus, a precise control of PLC-HDP functions is clearly critical for ordered development and homeostasis.
In concordance with their role as transcriptional regulators, homeoproteins localize predominantly to the nucleus, although several reports characterize them also as nucleocytoplasmic shuttle proteins, e.g., Extradenticle (1), Otx1 (56), and Engrailed (34). Since regulated subcellular localization has been reported for several transcription factors (e.g., p53, STATs, NF-
B, etc.) (6), we investigated the intracellular trafficking of PLC-HDPs and analyzed its consequences for PLC-HDP function as transcriptional regulators. As representatives of the Vsx-1 and Vsx-2 group, we studied the zebra fish Vsx1 and the murine Chx10 protein in detail. Nucleocytoplasmic transport was investigated by interspecies heterokaryon assays, microinjection of recombinant transport substrates, and the use of chemical transport inhibitors. We could demonstrate that PLC-HDPs contain a Crm1-dependent NES, previously described as the "octapeptide." Nuclear export influenced PLC-HDP transcriptional activation by enhancing proteasomal protein degradation and by facilitating extracellular secretion. The predominant nuclear steady-state localization of PLC-HDPs is mediated by the presence of an active nuclear import signal. This NLS can function also as a protein transduction domain (PTD), explaining the evolutionary conservation of this signal. The integrity of both NES and NLS/PTD appears to be prerequisite for PLC-HDPs to function as mobile nucleocytoplasmic shuttle proteins with the potential for intercellular transfer.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells, transfection, microscopy, and microinjection. Vero cells, the microglia cell line CRL-2540, 293 cells, NIH 3T3 cells, and HeLa cells were maintained under conditions recommended by the American Type Culture Collection and were prepared for microinjection or transfected as described previously (18). Microinjection, observation, and image analysis in living or fixed cells were performed as described previously (18). Cells were observed and analyzed by using the appropriate fluorescence filters as described previously (19), and 12-bit black and white images were captured by using a digital Axiocam CCD camera (Zeiss). Quantitation, image analysis, and presentation was performed by using IPLab Spectrum (Scanalytics) and Axiovision software (Zeiss). The total cellular GFP signal was measured by calculating the integrated pixel intensity in the imaged cell multiplied by the area of the cell. The nuclear signal was similarly obtained by measuring the pixel intensity in the nucleus. The cytoplasmic signal was calculated by subtracting the nuclear signal from the total cellular signal. All pixel values were measured below the saturation limits, and the background signal in an area with no cells was subtracted from all values. To determine the average intracellular localizations of the respective proteins, at least 200 fluorescent cells in three independent experiments were examined, and the standard deviations were determined.
Transactivation assays. For transactivation assays, HeLa cells were transfected with 0.5 µg of the pLCR-R-luc reporter plasmid and the indicated amounts of the MmChx10 expression constructs, together with 0.1 µg of pSV40-Gal, and the cells were assayed for luciferase and ß-galactosidase (ß-Gal) activity as described previously (23). To analyze intercellular transactivation, 5 x 105 293 cells were transfected with either 3 µg of the indicated MmChx10 expression construct or with 1 µg of pLCR-R-luc and 0.1 µg of pSV40-Gal. At 12 h later MmChx10 transfected and pLCR-R-luciferase transfected cells were mixed at a ratio of 2:1 and assayed for luciferase and ß-Gal activity 36 h later. Luciferase activity was normalized to ß-Gal expression, and all measurements were conducted in duplicates in three independent experiments.
Purification of recombinant GST fusion proteins. GST-GFP hybrid proteins were expressed and purified as described previously (45). Removal of GST by proteolytic cleavage using factor Xa protease (Roche) was performed according to the manufacturer's recommendations.
Immunoblotting, immunofluorescence, and antibodies. Immunoblotting and immunofluorescence were carried out according to standard procedures, as previously described (18). Purified mouse Chx10 fused to GST was used for immunization of rabbits by using standard protocols (10). The immunoglobulin G fraction was purified by protein A chromatography and used at a 1:500 dilution for immunofluorescence.
Protein transduction assay. Exponentially growing Vero cells were incubated with 1 µM concentrations of the corresponding recombinant GFP fusion proteins in phosphate-buffered saline (PBS) for 2 h. Subsequently, cells were extensively washed with PBS before incubation with trypsin (1 mg/ml) for 2 min to remove unspecifically bound protein from the cell surface. After removal of trypsin cells were cultured in medium for 3 h, washed with PBS, fixed with ice-cold methanol for 15 min and rehydrated in PBS prior to analysis by fluorescence microscopy.
Heterokaryon assay. HeLa cells were transfected with the indicated plasmids and 12 h later seeded with untransfected mouse NIH 3T3 cells at a ratio of 1:3. Cells were cultured and fused 8 h later by using polyethylene glycol (Gibco) in the presence of cycloheximide as described previously (50). To discriminate between human donor and mouse acceptor nuclei, staining with Hoechst 33258 was performed as described previously (50). A total of 50 heterokaryons were chosen at random, and the percentage of fusion events positive for internuclear transfer was calculated in three independent experiments, and the standard deviations were determined.
Treatment with chemical export inhibitors. Cells transfected with the indicated plasmids were treated with 10 nM leptomycin B (LMB; Sigma-Aldrich) or 5 nM Ratjadone A (Alexis Biochemicals) as described previously (25).
Crm1 pull-down assays and in vitro translation. Coupled transcription-translation was performed by using the TNT reticulocyte lysate system (Promega) supplemented with [35S]methionine (Amersham) and the plasmid p3-Crm1-HA as a template. Crm1 pull-down assays with the specific recombinant GST-GFP substrates, Ran-GTP and nuclear extracts were performed as described previously (18). Care was taken to ensure equal input levels of labeled Crm1 protein into the binding reactions.
Secretion assay. A total of 2 x 106 293 cells were transfected with the indicated plasmids and incubated for 8 h. Subsequently, cells were cultured in methionine-free medium supplemented with [35S] methionine (50 µCi) for additional 12 h. To block classical protein secretion, brefeldin A (BFA; Sigma-Aldrich) at 10 µg/ml was added to the cultures. Culture supernatants were collected and cleared by centrifugation (10,000 x g, 1 h, 4°C). Analysis of whole-cell lysates and immunoprecipitation of GFP fusion proteins from culture supernatants and cellular lysates by using a polyclonal anti-GFP antibody (BD Biosciences), as well as analysis of the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, were performed as described previously (18, 28).
Pulse-chase experiments. A total of 5 x 105 HeLa cells were transfected with pc3DrVsx1-GFP or pc3DrVsx1_NESmut-GFP, followed by incubation for 16 h. Subsequently, cells were incubated for 2 h in Dulbecco’s modified Eagle medium lacking methionine and pulse-labeled with 50 µCi of [35S]methionine (Amersham) for 2 h. Unlabeled methionine was then added to a final concentration of 100 mM. At the indicated time points, cells were washed with cold PBS, and whole-cell lysates were prepared as described previously (28). To prevent proteasomal degradation, cells were treated with the proteasome inhibitors MG-132 and hemin (Sigma-Aldrich; 50 µM final concentration). The total radioactivity in each sample was determined by trichloroacetic acid precipitation, and sample volumes were adjusted to represent equal amounts of radioactivity. Immunoprecipitation was done by using a polyclonal anti-GFP antibody (Clontech), and the complexes were resolved by SDS-PAGE as described previously (18). Band intensities were quantified by using a phosphorimager (Bio-Rad).
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RESULTS |
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To further address whether DrVsx1-GFP and MmChx10-GFP were capable of nucleocytoplasmic trafficking, we performed heterokaryon assays in the presence of cycloheximide to prevent de novo protein synthesis. Upon fusion of DrVsx1-GFP and MmChx10-GFP expressing HeLa donor cells with untransfected NIH 3T3 acceptor cells, both PLC-HDPs were exported from the donor and imported into the mouse acceptor nuclei 60 min after fusion (Fig. 1F and G). As a control, incubation of the fused cells at 4°C (data not shown) or in the presence of LMB did not result in detectable accumulation of GFP fusion proteins in the acceptor nuclei, indicative for active transport (Fig. 1G). Since the cytoplasm of donor and acceptor cells are fused in the heterokaryon assay the low amount of cytoplasmic DrVsx1-GFP or MmChx10-GFP protein, respectively, initially present in the donor cells was diluted and not detectable postfusion. Of note, the level of nuclear fluorescence in the acceptor nuclei increased, and the fluorescence signal in the donor nuclei decreased over time, excluding the formal possibility that the observed nuclear transfer events resulted from the import of the cytoplasmic GFP fusion proteins present prior to fusion.
PLC-HDPs contain a highly conserved NES previously described as the "octapeptide." To identify domains directing nuclear export, we first expressed N- and C-terminal deletion mutants of DrVsx1 and MmChx10 as GFP hybrids. As indicated in Fig. 1B, only fusion proteins containing the first 47 aa responded to LMB treatment, indicating the presence of an active NES. Database searches identified potential NES in the PLC-HDPs, matching the still loosely defined consensus sequence for leucine-rich NES (18, 20). Because predicted signals need to be verified experimentally, we tested the activity of the potential NES in a highly stringent system that allows the observation and quantification of nuclear export in living cells, independent of drug treatment, nuclear import, and passive diffusion (45). Signals (Fig. 2A) were expressed as fusions with GST and GFP (GST-NES-GFP) and tested by microinjection. Due to the size of the fusion proteins (54 kDa, as a monomer) the localization of the microinjected autofluorescent transport substrate is not flawed by passive diffusion, and the protein remains at the site of injection for up to 24 h (45). We observed that only substrates containing active PLC-HDP NES were quantitatively exported into the cytoplasm within 16 h after microinjection into the nucleus of Vero (Fig. 2B) and microglia CRL-2540 cells (data not shown). As a stringent control, a signal in which essential residues were replaced by alanines was inactive under identical experimental conditions (Fig. 2A and B). Likewise, treatment with LMB completely prevented export (data not shown). Interestingly, analysis of NES representative for all known PLC-HDP family members revealed that these NES mediated export with comparable kinetics (Fig. 2A and B). Approximately 100 cells were injected and analyzed, and representative examples are shown. These results were confirmed in two independent experiments (data not shown). The evolutionary conservation of the NES strongly argues that nuclear export is critical for the biological function of PLC-HDPs. Since nuclear export had been proposed also for other members of the homeoprotein family (34), we included the proposed NES of the Engrailed homeoprotein (Fig. 2A; GgEn2 NES) in our study. In contrast to the PLC-HDP NES, the GgEn2 NES was not active in our assay (data not shown).
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37AAITDLAGA45; MmChx10_NESmut-GFP, aa32FGIQEILGL40 32AGIQEIAGA40). In contrast to the wild-type proteins DrVsx1_NESmut-GFP and MmChx10_NESmut-GFP displayed a complete nuclear localization after transient transfection (Fig. 1C). Likewise, the NES-deficient mutants were not exported in the heterokaryon assays (Fig. 1F and G), excluding the presence of additional NES or the possibility that export was mediated by shuttling interaction partners in trans. PLC-HDP export signals interact with Crm1 in vitro. If the defined PLC-HDPs are exported via the Crm1 pathway, these proteins should interact with the export receptor also in a cell-free system. We therefore performed in vitro interaction assays to biochemically verify the Crm1 interaction. Figure 2C demonstrates that recombinant GST-MmChx10-GFP significantly bound to Crm1 in the presence of Ran-GTP and nuclear extracts in contrast to inactive GST-MmChx10_NESmut-GFP or GST-GFP alone. Similar results were obtained for GST-DrVsx1-GFP or the other GST-NES-GFP fusion proteins, respectively (data not shown).
Nuclear export facilitates intracellular degradation of DrVsx1-GFP. Kurtzman et al. (26) reported the polyubiquitination and degradation of DrVsx1 by the ubiquitin/proteasome pathway. Because the proteasome degradative pathway appears to operate predominantly in the cytoplasm (51), we investigated whether nuclear export influences indirectly the intracellular stability of DrVsx1. HeLa cells transiently expressing DrVsx1-GFP or DrVsx1_NESmut-GFP, respectively, were metabolically pulse-labeled, followed by a chase with cold methionine for 0, 30, 60, and 120 min. Subsequently, GFP fusion proteins were immunoprecipitated with anti-GFP antiserum and resolved by SDS-PAGE (Fig. 3A). Band intensities from two independent experiments were quantified by using a phosphorimager; this showed that both DrVsx1-GFP and DrVsx1_NESmut-GFP were degraded over time and that degradation could be reduced by treatment with proteasomal inhibitors. Interestingly, preventing nuclear export resulted in a significantly increased half-live for DrVsx1_NESmut-GFP (Fig. 3A and B), suggesting that nuclear export is continuously supplying substrate for the proteasomal degradation machinery. Similar results were obtained for MmChx10 (data not shown).
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Nuclear export facilitates unconventional secretion of PLC-HD proteins. Having demonstrated that PLC-HDPs are nucleocytoplasmic shuttle proteins, we investigated their potential for intercellular trafficking. In general, intercellular transfer requires both internalization and secretion. To analyze secretion of DrVsx1-GFP and to investigate the influence of nuclear export on secretion, we attempted to recover metabolically labeled DrVsx1-GFP or DrVsx1_NESmut-GFP protein, respectively, from the culture supernatant of transfected 293 cells. Figure 4A illustrates that DrVsx1-GFP, but not DrVsx1_NESmut-GFP could be immunoprecipitated by anti-GFP antibodies from the supernatant. Transfected cells were controlled by microscopic observation for cytotoxic effects caused by the expression of the respective GFP fusion proteins prior to lysate preparation to minimize unspecific protein release due to cell death. To also exclude the possibility that the observed result reflects differences in protein expression, equal expression levels of the GFP fusion proteins were verified by Western blot analysis of cellular extracts (Fig. 4B). Similar results were obtained for the MmChx10-GFP or MmChx10_NESmut-GFP protein, respectively (data not shown). Of note, we could not recover a NES-GFP fusion protein (DrVsx1 NES-GFP) from the supernatant of transfected 293 cells (data not shown). Thus, the continuous supply of cytoplasmic DrVsx1 from the nuclear pool by active nuclear export appears to facilitate secretion, but the NES itself does not represent an unconventional secretion signal. To determine whether protein secretion was mediated via the classical endoplasmic reticulum/Golgi-dependent pathway or by unconventional secretion, we attempted to inhibit secretion by treatment of the transfected cells with BFA. However, BFA treatment did not interfere with MmChx10-GFP release in support of secretion by the unconventional pathway, as also reported for several other proteins (39; data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As transcription factors, PLC-HDPs have to access the nucleus to execute their function. Theoretically, the size of ca. 34 kDa allows PLC-HDPs to enter the nucleus also by passive diffusion. However, even smaller proteins are transported by active, signal-mediated mechanisms, most likely because active transport is more efficient and amendable to specific control mechanisms (3, 13). The transfection and/or microinjection experiments indicated that the conserved KRKKRRHR motif can not only function as a nuclear retention signal but also represents a bona fide monopartite nuclear import signal for PLC-HDPs in which the underlined arginines are critical for function and efficiency. Although Ubc9 has been suggested to mediate the nuclear localization of Vsx1 (27), we are currently investigating in detail whether Vsx1 is directly imported via the transportin 13/Ubc9 axis (37) or can also use alternative import pathways. Although the PLC-HDPs NLS is less active compared to the classical SV40 NLS (45), the rate of import still exceeds the rate of export, resulting in the observed dynamic but predominantly nuclear steady-state localization of PLC-HDPs.
The activity of transcriptional regulators can be modulated at various levels. As shown for other transcription factors (6, 44), these include posttranscriptional modifications in the nucleus or the cytoplasm, e.g., phosphorylation (35), sumoylation (10), and interactions with other proteins (14). The detailed molecular pathways regulating the activity of PLC-HDPs are currently under intense investigation. Here, we provided evidence that PLC-HDPs are dynamic transcription factors that have the capability to shuttle between the nucleus and the cytoplasm. Consequently, PLC-HDPs might be subjected to regulatory control mechanism homing in these specific compartments. Degradation by the ubiquitin/proteasome pathway is crucial to control protein homeostasis and was described for several transcription factors, including the DrVsx1 protein (26). In the present study, we found that inactivation of the nuclear export activity of DrVsx1 and MmChx10 resulted in increased protein stability and thus in increased transcriptional activation. Since the ubiquitin/proteasome pathway appears to act predominantly in the cytoplasm (47), our transactivation result indicate that regulating nucleocytoplasmic transport can indirectly influence the intracellular protein levels and the biological activity of PLC-HDPs by the proteasome pathway. A similar model was proposed for the IB/NF-B axis in which the nuclear export activity of IB regulates the intracellular localization, degradation, and transcriptional activity of NF-B (see references 30 and 33 and references therein). In contrast, Rehberg et al. reported that the inactivation of the NES in the Sox10 protein resulted in decreased transactivation by an unknown mechanism (44). Although it is less likely, we cannot formally rule out the possibility that the mutations introduced to generate the export deficient DrVsx1 protein directly resulted in enhanced degradation resistance. The increased stability observed would thus be due to conformational changes and not caused by blocking export.
We found that the continuous supply of cytoplasmic DrVsx1 or MmChx10 from the nuclear pool by active export facilitated also extracellular release of the proteins. PLC-HDPs in general lack a canonical secretion signal and appear not to be targeted into the endoplasmic reticulum by a cotranslational mechanism. In addition, secretion could not be inhibited by treatment with BFA, an inhibitor of the classical endoplasmic reticulum/Golgi-dependent secretion pathway. Thus, extracellular release appears to be mediated by the unconventional secretion pathway reported also for several viral and cellular proteins (see reference 39 and references therein). In this context, Julian Huxley's term "growth gradient" may be relevant for the biological function of PLC-HDPs. In the morphogen gradient model, the local concentration of a diffusible molecule can determine cells' rates of proliferation and differentiation as a continuous function of concentration (4, 11, 36). This model is made particularly attractive by our finding that PLC-HDPs also harbor a highly conserved PTD with a similar activity as the widely used HIV-1 Tat PTD (52). The mechanism of transduction has been studied extensively for a variety of proteins, including the Antennapedia and PDX-1 homeodomain transcription factors (15). Recent evidence suggests that transduction occurs via a multistep mechanism involving endocytosis and macropinocytosis (54). Although we could not visually monitor the spread of DrVsx1-GFP or MmChx10-GFP from expressing donor to untransfected acceptor cells as described for the Engrailed protein (22), the results of our intercellular transactivation assays support the intercellular trafficking of PLC-HDPs, as demonstrated for other PTD-containing transcription factors (53). However, to fully understand the biological relevance of transduction in vivo, the activity of PTD-deficient PLC-HDP mutants has to be investigated in adequate animal models.
In summary, our report provides novel insights into the functional domain organization of PLC-HDPs (Fig. 7A). Based on our findings, we propose a model in which the continuous nucleocytoplasmic shuttling of PLC-HDPs contributes to the optimal and flexible execution of their transcriptional activities (Fig. 7B). The evolutionarily conserved combination of a PTD, together with active nuclear export and import signals, may allow the fine-tuning of intracellular protein levels by the proteasome pathway and, in addition, also permits intercellular transfer. The overlapping complex patterns of homeobox gene expression in the embryonic retina requires a complex regulatory network of transcription factors that specifies differentiation of competent retinal progenitors. Although transcriptional regulation of PLC-HDPs is an important control mechanism, PLC-HDPs may have additional unexpected paracrine activity, thereby influencing and maintaining the complex expression pattern during development. To ultimately gain profound insight into the detailed role of PLC-HDPs' nucleocytoplasmic shuttling for ordered development has to await transgenic mouse knock-in models in which nucleocytoplasmic transport of Chx10 is selectively abolished. Currently, we are pursuing this strategy in our laboratory.
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ACKNOWLEDGMENTS |
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This study was supported by the Deutsche Forschungsgemeinschaft (Sta 598/1-2) and the Studienstiftung des Deutschen Volkes (S.K.K.).
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FOOTNOTES |
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), DrVsx1-GFP + proteasomal inhibitors (PI) (•), DrVsx-1_NESmut-GFP (
), and DrVsx-1_NESmut-GFP + PI (
). (C) Luciferase assays after cotransfection of HeLa cells with pSV40-Gal, an MmChx10-responsive luciferase reporter and different amounts of expression plasmids for GFP, MmChx10-GFP, and MmChx10_NESmut. Expression of wild-type MmChx10-GFP resulted in higher transcriptional activation compared to the export-deficient mutant. Luciferase activity was normalized to ß-Gal expression. Error bars indicate the standard deviations.
