Green fluorescent protein-tagging reduces the nucleocytoplasmic shuttling specifically of unphosphorylated STAT1 Thomas Meyer 1 , Andreas Begitt 2 and Uwe Vinkemeier 2 1 Abteilung Psychosomatische Medizin und Psychotherapie, Philipps-Universita ¨ t Marburg, Germany 2 Abteilung Zellula ¨ re Signalverarbeitung, Leibniz-Institut fu ¨ r Molekulare Pharmakologie, and Freie Universita ¨ t Berlin, Germany Cytokines and growth factors modulate the trans- criptional activity of their target cells. To this effect, cytokine-specific signal transducers of the signal trans- ducers and activators of transcription (STAT) family of transcription factors are activated at membrane- bound receptors and subsequently translocate into the nucleus [1,2]. ‘Activation’ is a multistep process leading to the tyrosine phosphorylation and subsequent dime- rization of STATs that enables high affinity and sequence-specific recognition of DNA [3–5]. In the nucleus, the activated STAT dimers stimulate or repress gene transcription by directly binding to Keywords FRAP; GFP; nuclear export; nuclear import; STAT1 Correspondence U. Vinkemeier, Abteilung Zellula ¨ re Signalverarbeitung, Leibniz-Institut fu ¨ r Molekulare Pharmakologie, FU Berlin, Robert-Ro ¨ ssle-Str. 10, 13125 Berlin, Germany Fax: +49 30 94793 179 Tel: +49 30 94793 171 E-mail: vinkemeier@fmp-berlin.de (Received 21 August 2006, revised 1 December 2006, accepted 6 December 2006) doi:10.1111/j.1742-4658.2006.05626.x Fluorescence recovery after photobleaching (FRAP) and related techniques using green fluorescent protein (GFP)-tagged proteins are widely used to study the subcellular trafficking of proteins. It was concluded from these experiments that the cytokine-induced nuclear import of tyrosine-phos- phorylated (activated) signal transducer and activator of transcription 1 (STAT1) was rapid, while the constitutive shuttling of unphosphorylated STAT1 was determined to be inefficient. However, unrelated experiments came to different conclusions concerning the constitutive translocation of STAT1. Because these discrepancies have not been resolved, it remained unclear whether or not unphosphorylated STAT1 is a relevant regulator of cytokine-dependent gene expression. This study was initiated to examine the influence of GFP-tagging on the nucleocytoplasmic shuttling of phos- phorylated and unphosphorylated STAT1. In accordance with previous findings our results confirm the undisturbed rapid nuclear import of GFP- tagged activated STAT1. However, we reveal an inhibitory influence of GFP specifically on the constitutive nucleocytoplasmic cycling of the unphosphorylated protein. The decreased shuttling of unphosphorylated STAT1-GFP significantly reduced the activation level while nuclear accu- mulation was prolonged. Importantly, despite unimpaired nuclear import of activated STAT1 the transcription of a STAT1-dependent reporter gene was more than halved after GFP-tagging, which could be linked directly to reduced nucleocytoplasmic shuttling. In conclusion, it is demonstrated that GFP-based techniques considerably underestimate the actual shuttling rate of unphosphorylated native STAT1. The results confirm that the activation of STAT1 and hence its transcriptional activity is proportional to the nucleocytoplasmic shuttling rate of the unphosphorylated protein. More- over, our data indicate that GFP-tagging may differently affect the mechanistically distinct translocation pathways of a shuttling protein. Abbreviations FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; IFN, interferon; LMB, leptomycin B; NES, nuclear export signal; STAT, signal transducer and activator of transcription. FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS 815 cognate binding sites in the promoters of cytokine- responsive genes [6]. By light microscopy this sequence of events is observable and appears as the nuclear accumulation of STAT molecules within minutes of cy- tokine stimulation [7]. The accumulation phase can last for up to a few hours before the prestimulation distri- bution is gradually restored. Initially, the STATs were considered cytoplasmic proteins that enter the nuclear compartment only in response to cytokine stimulation. This simple concept has been amended recently and a more variegated model of STAT functioning was pro- posed [8]. In the revised model the STATs are des- cribed as nucleocytoplasmic shuttling proteins that enter the nucleus irrespective of cytokine stimulation or tyrosine phosphorylation. The constitutive import activity of STAT1 occurs independent of transport receptors (also termed karyopherins) via direct con- tacts with nucleoporins in the nuclear pore. The same mechanism applies also for nuclear export, although carrier-dependent transport via CRM1 (also known as exportin 1) contributes [9]. Activated STAT1, however, is barred from further karyopherin-independent shut- tling [9,10]. Yet, dimerization triggers the exposure of noncanonical import signals, and the concomitant binding of transport receptors results in nuclear import [11–13]. Nuclear export of STAT1, on the other hand, requires its tyrosine dephosphorylation [10,14]. The possibility to use genetically encoded fluorescent markers such as green fluorescent protein (GFP) in combination with laser bleaching has allowed to directly follow the redistribution of molecules in living cells [15,16]. This approach was used to study the intracellular trafficking of STAT1, and no significant differences were reported between tagged and endo- genous native molecules [17–19]. These experiments demonstrated that the inducible nuclear translocation of phosphorylated STAT1-GFP caused the rapid nuc- lear import and accumulation, whereas the constitutive nucleocytoplasmic shuttling of unphosphorylated STAT1-GFP in unstimulated cells occurred at a very low rate. It was thus concluded that these results were applicable also to the endogenous native STAT1. Yet, unrelated experiments and theoretical considerations indicated that efficient shuttling also of unphosphoryl- ated STATs is required for efficient cytokine signalling [10,20,21]. These discrepancies have not been resolved to date. Here, we analyze the influence of GFP-tagging on phosphorylated and unphosphorylated STAT1. In accordance with previous results we find that the trans- port of phosphorylated STAT1 was not affected by the GFP domain. Contrary, the nucleocytoplasmic shut- tling of unphosphorylated STAT1 was considerably reduced after GFP-tagging. Accordingly, the phos- phorylation and DNA binding decayed significantly faster. Moreover, the reduced translocation of unphos- phorylated STAT1-GFP diminished the transcription of a reporter gene by 60%, even though the nuclear accumulation was prolonged. Thus, these analyses demonstrate that GFP-based techniques considerably underestimate the actual shuttling rate of unphosphory- lated native STAT1. The results confirm the functional importance of efficient translocation of nonphosphory- lated STAT1 for the cytokine-dependent transcription. Results GFP-tagging prolonged the duration of STAT1 nuclear accumulation, but not its build-up phase Carboxy-terminal fusion proteins of STAT1 with GFP are generally regarded to behave indistinguishably from the endogenous wild-type protein. At first, we compared the accumulation kinetics of wild-type STAT1 and GFP-tagged STAT1 transcription factors upon stimulation of cells with interferon c (IFNc). Interferon induces the transient accumulation of STAT1 molecules within the nuclear compartment, which lasts for a few hours before the prestimulation distribution is restored [1]. As previously reported [17], we found that the nuclear import rate of STAT1 is independent of the expression of the GFP domain in IFNc-stimulated cells, because the build-up of nuclear accumulation did not differ between the native and the GFP-tagged variant. Already 5 min after exposure of the cells to IFNc both native STAT1 in untransfected cells and STAT1-GFP in cells expressing the GFP fusion lost their predominantly cytoplasmic resting dis- tribution (Fig. 1A, panels a,g) and began to accumu- late in the nucleus (Fig. 1A, panels b,h). Ten minutes after IFNc addition to the cells, STAT1 is concentra- ted within the nucleus, irrespective of the expression of the GFP domain (Fig. 1A, panels c,i). After 60 min exposure to IFN c the cytoplasm appeared depleted of either STAT1 immunoreactivity or GFP fluorescence, indicating that the cytokine-induced nuclear import of tyrosine-phosphorylated STAT1 is not protracted by the expression of the GFP fusion (Fig. 1A, panels d,j). In contrast to the build-up of nuclear accumulation, the decay of STAT1 accumulation in the nucleus dif- fered considerably between the tagged and untagged variants. Four hours after a one-hour stimulation with IFNc, the accumulation phase of native STAT1 was nearly over (Fig. 1A, panel e) and an additional two hours later the distribution of unstimulated cells was regained (Fig. 1A, panel f). GFP-tagged STAT1 GFP-tagging of STAT1 T. Meyer et al. 816 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS protein, however, remained largely nuclear also four hours after the initial IFNc stimulation (Fig. 1A, panel k). Even after another two hours STAT1-GFP was still predominantly nuclear (Fig. 1A, panel l). Pro- longed nuclear accumulation of STAT1-GFP was seen not only with HeLa cells, but also with 293T cells or 2fTGH cells (not shown). In addition, we transfected STAT1-deficient U3A cells with wild-type or GFP- tagged STAT1. Subsequently, the cells were fixed and stained identically with a STAT1 antibody to observe the nucleocytoplasmic translocation of both STAT1 variant proteins by indirect immunofluorescence micro- scopy (Fig. 1B). As indicated by the staining intensi- ties, both STAT1 variant proteins were expressed at comparable levels. Moreover, as seen before in HeLa cells (Fig. 1A), also in U3A cells the nuclear accumula- tion of untagged STAT1 was terminated sooner. Wild- type STAT1 accumulated in the nucleus only for about one hour after interferon stimulation (Fig. 1B, panels b,c). For GFP-tagged STAT1, however, the accumulation phase lasted about twice as long (Fig. 1B, panels i–k), before a pancellular distribution was reached (Fig. 1B, panel l). Taken together, our results confirm that the nuclear entry of activated STAT1 did not differ between native and GFP-tagged STAT1. The duration of nuclear accumulation, on the other hand, was markedly prolonged by the fusion to GFP. The prolonged nuclear accumulation of GFP-tagged STAT1 was associated with reduced tyrosine phosphorylation and DNA binding Because only unphosphorylated STAT1 can exit the nucleus, the prolonged nuclear accumulation of STAT1-GFP may be caused by defective tyrosine dephosphorylation. This was examined in HeLa cells transiently expressing STAT1-GFP. As can be seen Fig. 1. GFP-tagging results in a prolonged nuclear accumulation of STAT1. (A) Shown is the time course of nuclear accumulation of either endogenous or GFP-tagged STAT1 in interferon (IFN) c-stimulated HeLa cells, as determined by immunocytochemistry using a STAT1-speci- fic antibody (left) and direct fluorescence microscopy (right), respectively. Cells were either left untreated (a, g) or treated with 5 ngÆmL )1 IFNc for 5 min (b, h), 10 min (c, i) or 60 min (d, j) before fixation. In (e, f, k, l) the cells were pretreated with IFNc for 60 min followed by incubation for additional 4 h (e, k) or 6 h (f, l) in IFNc-free medium. Shown are fluorescence micrographs demonstrating the intracellular local- ization of STAT1 and STAT1-GFP, respectively, and the corresponding Hoechst-stained nuclei. Note the rapid build-up of both endogenous and GFP-tagged STAT1 within the nucleus, which occurred with similar kinetics, and the delayed nuclear export of STAT1-GFP as compared to the wild-type protein. (B) STAT1-deficient U3A cells transiently expressing wild-type STAT1 (left) or STAT1-GFP (right) were left untreated (a, g) or stimulated with IFNc for 60 min. Subsequently, the cells were fixed and stained right away (b, h), or the cells were fixed after con- tinued incubation in the absence of interferon for the times indicated. Shown are indirect immunofluorescence results with a STAT1-specific antibody. T. Meyer et al. GFP-tagging of STAT1 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS 817 from the western blot of Fig. 2A (lower panel), the expression levels of endogenous STAT1 and recom- binant STAT1-GFP were comparable. Stimulation of the cells with IFNc induced the comparable tyrosine phosphorylation of native and tagged STAT1. How- ever, the phosphorylation signal decayed faster for GFP-tagged STAT1 (Fig. 2A). A quantitative analysis of the western blotting results clearly demonstrated this outcome (Fig. 2B). Because high-affinity DNA binding requires the tyrosine phosphorylation of STAT1, we also analyzed the time course of DNA binding. As is shown in Fig. 2C, endogenous and STAT1-GFP bound to DNA predominantly as ho- modimers, although a minor proportion of hetero- dimers was also observed (Fig. 2C, asterisk). As expected from the time course of STAT1 activation shown in Fig. 2A, the DNA binding activity of STAT1-GFP decayed faster than the DNA binding of the endogenous STAT1 (see the quantitation, Fig. 2D). We concluded that the fusion of STAT1 with GFP did not impair the DNA binding and the tyrosine dephosphorylation reaction. Hence, the pro- longed nuclear presence of STAT1-GFP was not due to enhanced activation, but the result of nuclear retention of unphosphorylated molecules. Protein microinjection and pharmacological export inhibition revealed that GFP-tagging impairs the nucleocytoplasmic translocation specifically of unphosphorylated STAT1 To directly analyze the influence of GFP-tagging on the nucleocytoplasmic translocation of STAT1, we per- formed microinjection studies with HeLa cells using recombinant STAT1 and STAT1-GFP purified from baculovirus-infected insect cells. Co-injected chromo- phor-labelled bovine serum albumin failed to cross the nuclear envelope of injected cells and thus served as a AB D C Fig. 2. Kinetics of tyrosine phosphorylation (A, B) and DNA binding activity (C, D) of untagged and GFP-tagged STAT1. Equal numbers of HeLa cells coexpressing endogenous STAT1 and the recombinant STAT1-GFP (Fig. 1A) were either left untreated (– IFNc) or stimulated with IFNc for the indicated times (+ IFNc, 1–4 h). (A) Shown is a western blot of whole cell extracts with a phospho-specific STAT1-Tyr701 anti- body (upper), and a reprobing with anti-STAT1 IgG (lower). The positions of native STAT1 (lower mark) and the GFP fusion (upper mark) are indicated. In (B) the normalized signal intensities for tyrosine-phosphorylated STAT1 [(signal Tyr-phosphorylated STAT1) ⁄ (signal unphosphoryl- ated STAT1)] of three independent experiments were densitometrically quantified and plotted. (C, D) Protein extracts prepared from the same cells as described in (A) were subjected to DNA binding analysis. Extracts were incubated with radiolabelled M67 probe and separated on 4% nondenaturing polyacrylamide gels. The positions of homodimers of endogenous STAT1 (lower mark), of homodimers of recombinant STAT1-GFP (upper mark), and of heterodimers thereof (asterisk) are indicated at the right margin of the gel. The arrowhead marks an unspe- cific band. Included are whole cell extracts from STAT1-negative U3A cells (lane 1) and two supershift experiment with HeLa cell extracts in the presence of anti-GFP (lane 2) and anti-STAT1 IgG (lane 3), respectively. Results from quantification of three independent experiments are shown in (D). GFP-tagging of STAT1 T. Meyer et al. 818 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS marker for the injection site. At first, STAT1 and STAT1-GFP were injected in the cytoplasm, followed by the stimulation of cells with IFNc for 30 min or 60 min to trigger tyrosine phosphorylation. Thereafter, cells were fixed and processed for immunocytochemis- try (wild-type), or the GFP epifluorescence was observed directly (STAT1-GFP). As is shown in panels A–D of Fig. 3, both STAT1 variants rapidly accumulated in the nucleus, irrespective of the GFP domain. This result confirms earlier reports of unper- turbed nuclear import of activated STAT1-GFP [17,18]. Fig. 3. Microinjection of purified, recombin- ant STAT1 reveals impaired nucleocytoplas- mic translocation of GFP-tagged STAT1. Unphosphorylated wild-type STAT1 or the GFP fusion thereof were purified from bacu- lovirus-infected Sf9 cells and injected at con- centrations of 1 mgÆmL )1 into either the cytosol (A–J) or the nucleus (K–P) of resting HeLa cells grown on poly L-lysine-coated glass coverslips. As a marker for the injec- tion site 0.2 mgÆmL )1 tetramethylrhodamine isothiocyanate- (T-) or fluorescein isothiocya- nate-labelled bovine serum albumin (F-BSA) was coinjected. In (A–D) the injected cells were stimulated with 5 ngÆmL )1 IFNc and in (E–P) the cells were left untreated. At the indicated times after injection the cells were fixed and stained with anti-STAT1 IgG C-24 (A, B, E–G, K–M) or the GFP fluorescence was observed directly (C, D, H–J, N–P). For each condition about 20 cells were success- fully microinjected, one of which is shown. T. Meyer et al. GFP-tagging of STAT1 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS 819 Next, we examined the impact of the GFP domain on the nuclear import of unphosphorylated STAT1 (Fig. 3E–J). The experimental set up was the same as in panels A–D, but the stimulation of cells with interferon was omitted. As expected, unphosphorylated STAT1 rapidly entered the nucleus, leading to a nearly pancel- lular distribution already after 30 min (Fig. 3E) that did not change much for another 90 min (Fig. 3F,G). GFP-tagged STAT, however, had a reduced rate of nuclear import. After 30 min only a small amount of the injected GFP variant was detectable in the nucleus (Fig. 3H), and even after another 30 min or 90 min the concentration of STAT1-GFP remained lower in the nucleus than in the cytoplasm (Fig. 3I,J). Similar results were obtained for the nuclear export of unphosphorylated STAT1. Wild-type or GFP- tagged STAT1 was microinjected into the nuclei of resting cells and the appearance of STAT1 in the cyto- plasm was monitored (Fig. 3K–P). As early as 15 min after nuclear delivery of STAT1, significant amounts of the wild-type protein were detected outside of the nucleus (Fig. 3K) and after 30–60 min the pancellular distribution was reached (Fig. 3L,M). In contrast, nuc- lear export of the GFP fusion protein was significantly reduced, as it took more than 30 min to detect measur- able fluorescence signals outside the injected nucleus (Fig. 3N,O). Even after 60 min the majority of the injected STAT1-GFP was still restricted to the nucleus, indicating that the fusion to GFP had diminished the nuclear export rate (Fig. 3P). The microinjection studies with purified exogenous recombinant proteins shown in Fig. 3 indicated that import of GFP-tagged STAT1 was reduced in unstimu- lated cells. We wanted to confirm this conclusion also for endogenous STAT1 variants expressed in transfected cells. However, two obstacles complicate the analysis of STAT1 nuclear import in unstimulated cells. Most importantly, there are no drugs available to induce or inhibit the constitutive cytokine-independent nucleocy- toplasmic transport of STAT1. Additionally, STAT1 is only slightly more concentrated in the cytoplasm of un- stimulated cells, making it difficult to clearly discern changes in the nucleocytoplasmic distribution. In order to overcome these limitations, we placed a canonical nuclear export signal (NES) at the C-terminus of STAT1, thus generating STAT1-NES and STAT1-NES- GFP. Due to their increased nuclear export rates both NES mutants displayed an exclusively cytoplasmic localization in resting HeLa cells (Fig. 4A,D). Notably, the incubation with leptomycin B (LMB) rapidly Fig. 4. Analysis of the nuclear import of unphosphorylated STAT1. Shown is the distribution of STAT1 that expresses a nuclear export signal (NES) alone (STAT1-NES; A–C) or in combination with GFP (STAT1-NES-GFP; D–F). Untreated cells (A, D) display the cytoplasmic accumula- tion of both STAT1 variant proteins. Inactivation of the NES receptor CRM1 with leptomycin B (LMB) for 60 min (B, E) or 120 min (C, F) results in the nuclear translocation of STAT1. The STAT1 distribution was detected immunocytochemically in fixed HeLa cells by using anti- STAT1 IgG C-24 and an appropriate Cy3-conjugated secondary antibody. Note that LMB treatment did not result in the accumulation of STAT1 in the nucleus, thus indicating continued CRM1-independent nuclear export. In Figs 4 and 5 the exposure time was reduced to sup- press detection of the endogenous STAT1 (see Experimental procedures). GFP-tagging of STAT1 T. Meyer et al. 820 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS incapacitates the NES receptor CRM1 [22]. This, in turn, triggers the collapse of STAT1-NES cytoplasmic accumulation. Thus, we generated an inducible system to analyze the influence of GFP-tagging on the cyto- kine-independent nuclear import of STAT1 (Fig. 4). Cells expressing STAT1-NES or STAT1-NES-GFP were fixed one or two hours after the addition of LMB, and STAT1 was detected immunocytochemically. In accordance with the protein microinjection results shown in Fig. 3, we found protracted nuclear import of GFP-tagged STAT1 also in transfected cells. While the GFP fusion protein was still predominantly cytoplasmic one hour after the addition of LMB (Fig. 4E), the un- tagged STAT1-NES had already reached the pancellular distribution at this time point (Fig. 4B). Conversely, the NES-variants of STAT1 can also be used to analyze nuclear export. For this purpose cells expressing the NES mutants were treated with IFNc in addition to LMB, which resulted in their comparable nuclear accumulation after 60 min (Fig. 5B,F). The cells were then incubated for another two or four hours in the presence of LMB, before the cells were fixed and immunostained for STAT1. As was seen in Fig. 1A and 1B for the endogenous and transfected wild-type protein in comparison to STAT1-GFP, also STAT1-NES returned to the cytoplasm much more quickly than the respective GFP-tagged variant. The non-GFP-tagged STAT1-NES displayed a pancellular distribution after 4 h (Fig. 5D), whereas the respective GFP-tagged mutant was still predominantly nuclear at this time point (Fig. 5H). Taken together, the results of Figs 3–5 demonstrate the inhibitory influence of GFP-tagging on both the nuclear import and export specifically of unphosphorylated STAT1. Based on the time required to achieve pancellular distribution, it appeared that the translocation rates were reduced at least by a factor of 2. GFP-tagging decreases the transcriptional activity of STAT1 by reducing its nucleocytoplasmic shuttling We have demonstrated here that nucleocytoplasmic shuttling of GFP-tagged STAT1 is slow in comparison to native STAT1. Hence the cytokine sensitivity of STAT1-GFP was reduced accordingly (Fig. 2). Finally, Fig. 5. Analysis of the nuclear export of unphosphorylated STAT1. HeLa cells expressing STAT1-NES (A–D) or STAT1-NES-GFP (E–H) were left unstimulated or stimulated for 60 min with 5 ngÆmL )1 IFNc and 10 ngÆmL )1 LMB to induce nuclear accumulation. Cells were then fixed immediately (A, B, E, F), or the incubation was continued for 2 h (C, G) or 4 h (D, H) in IFNc-free medium. Detection of STAT1 localization was by immunocytochemistry, and the nuclei were stained with Hoechst dye. Note the indiscriminate nuclear accumulation resulting from IFNc and leptomycin B treatment, and the slow return to the cytoplasm of the GFP-tagged variant. T. Meyer et al. GFP-tagging of STAT1 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS 821 we therefore examined the influence of the GFP fusion on the STAT1-dependent activation of a luciferase reporter gene. As shown in Fig. 6A, the gene induction by wild-type STAT1 was more than twice as efficient as by the GFP fusion protein, despite similar expres- sion levels of both proteins. Because GFP was fused to the C-terminal transactivation domain of STAT1, tran- scription could be compromised not only by the effects of GFP on nucleocytoplasmic shuttling, but also by reduced recruitment of transcription cofactors due to the presence of additional residues close to the tran- scription activation domain. In order to discriminate between these possibilities, we included in this analysis the mutant STAT1-NES. As we have demonstrated previously, the addition of a nuclear export signal to STAT1 resulted in comparable nuclear export rates ([21], see also Fig. 4) and hence minimized the activa- tion differences among STAT1 variant proteins [21]. A C B Fig. 6. Diminished nucleocytoplasmic shuttling of STAT1 is associated with a decrease in transcriptional activity. (A) STAT1-negative U3A cells were cotransfected with plasmids coding for either wild-type STAT1 or STAT1-GFP together with an IFNc-inducible luciferase reporter gene construct and a plasmid for constitutive expression of b-galactosidase. Luciferase activity was determined 24 h post-transfection in unstimulated cells (grey bars) or after stimulation with IFNc for 6 h (black bars). The luciferase expression induced by STAT1 and STAT1- NES were set to 100%. Error bars represent standard deviations for six independent experiments normalized to the expression of b-galac- tosidase. (B) Gel shift analysis with wild-type STAT1, STAT1-NES and STAT1-NES-GFP and a M67 STAT1-binding site. U3A cells expressing the STAT1 variant proteins were treated with IFNc for 60 min or left unstimulated. Soluble proteins were extracted sequentially from the cytosol and the nucleus, and the combined extracts were used for further analyses. Extracts from IFN-stimulated cells were analyzed first by western blotting to determine the concentration of activated STAT1 (not shown). Subsequently, the IFN-treated extracts were normalized (see Experimental procedures), such that an identical quantity of each activated STAT1 variant was incubated with the radioactive DNA probe. Shown is the autoradiogram of the native PAGE. (C) U3A cells were cotransfected with plasmids encoding STAT1-NES or STAT1- NES-GFP and reporter genes as described in (A). The cells were stimulated with IFNc in the absence (black bars) or presence (grey bars) of leptomycin B. Gene induction in unstimulated cells was barely detectable both without (white bars) or with (not shown) leptomycin. The reporter gene activity was analysed as described (A). The inserts in (A) and (C) show western blotting results of whole cell extracts prepared from cells used for the reporter gene analyses. The extracts were probed with a pan-STAT1 antibody (top) and reprobed with an antibody against b-actin (bottom). GFP-tagging of STAT1 T. Meyer et al. 822 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS Moreover, the addition of the export signal did not diminish DNA binding of STAT1 (Fig. 6B) Remark- ably, however, when the export differences were equal- ized by the inclusion of the export signal, the expression of the GFP domain no longer reduced the transcriptional activity of STAT1 (Fig. 6C). In the presence of LMB, which specifically inactivates the exportin CRM1 and hence blocks the export enhance- ment conferred by the NES (Fig. 4), the transcriptional activity of GFP-tagged STAT1 again reached only about 50% of the untagged STAT1 (Fig. 6C). We therefore concluded that the observed inhibitory effect of GFP on gene transcription could be attributed primarily to its effects on STAT1 nucleocytoplasmic shuttling. Discussion The results presented here demonstrated that the nucleo- cytoplasmic translocation of the STAT1-GFP fusion protein differed from the endogenous wild-type protein. Our data indicate an inhibitory influence of GFP-tag- ging specifically on the constitutive nucleocytoplasmic shuttling of unphosphorylated STAT1. We used several independent experimental approaches to exclude stain- ing differences and overexpression artifacts as the cause of the reduced nucleocytoplasmic shuttling. We used C-terminally GFP-tagged STAT1 in our experiments, which was reported before to be indistinguishable from the wild-type protein [17,18,23]. However, only the ini- tial phases of the cytokine-induced nuclear accumula- tion were considered in those studies. Indeed, our data confirm that the cytokine-induced nuclear import of activated STAT1 and the onset of nuclear accumulation are not disturbed by the presence of the GFP domain at the STAT1 C-terminus. Yet, the nuclear accumulation of STATs is highly dynamic and dependent on their continuous nuclear export with subsequent rephosph- orylation at the cell membrane. Accordingly, the acti- vation level is immediately reduced when the shuttling rate falls. Consistent with this model, impaired cycling caused by GFP-tagging resulted in diminished activation and DNA binding. As expec- ted, this also adversely affected the transcriptional activity. While addition of the GFP domain to the STAT1 C-terminal transactivation domain did not inhibit transcriptional activation during stimulation of cells with IFN c per se, the inefficient cycling never- theless resulted in a strongly decreased transcriptional yield. These results underscore the physiological importance of continuous nucleocytoplasmic cycling for the cytokine-dependent gene regulatory functions of STAT1. GFP-tagging is used extensively to examine the intracellular mobility and nucleocytoplasmic shuttling of proteins. For STAT1, some of these studies indicate the relatively slow shuttling before the stimulation of cells with cytokines, and the rapid nuclear import of tyrosine-phosphorylated molecules during cytokine sti- mulation leading to nuclear accumulation [17–19]. The results presented in this work urge caution concerning the assignment of results that were obtained with GFP-tagged STAT1 to the native protein. Our data indicate that the constitutive shuttling rates of STAT1- GFP were reduced at least by a factor of two, while the nuclear import of activated STAT1-GFP was not measurably affected. We cannot conclude from our data that the constitutive and cytokine-induced trans- locations of native STAT1 occur with identical rates. However, the results presented here indicate that the translocation rates of STAT1 before and after cytokine stimulation are much more similar than suggested by fluorescence recovery after photobleaching (FRAP) analyses with GFP-tagged molecules. This conclusion is supported also by experiments performed with a STAT1 mutant that has lost the ability to interact with DNA [10]. Although there was no obvious difference in the nuclear import of the activated mutant in com- parison to wild-type STAT1, there was nevertheless no clearly discernible nuclear accumulation [10]. However, if nuclear import was stimulated substantially by IFN treatment whereas the constitutive shuttling remained at a slow rate, the outcome should be nuclear accumu- lation. Because this is not the case, increased nuclear import is unlikely to account for the transient cyto- kine-induced accumulation of STAT1 in the nucleus. Rather, the nuclear retention of activated molecules appears to play a prominent role. We thus conclude that translocation analyses by photobleaching tech- niques in combination with GFP-tagging do not properly reflect the dynamic redistribution of unphos- phorylated wild-type STAT1, because the nuclear envelope represents a major diffusion barrier for the constitutive cycling of STAT1-GFP. Photobleaching techniques, however, are well suited to examine the mobility of STAT1 in the cytoplasm or the nucleo- plasm, because the STAT1-GFP mobility, with or without ligand stimulation, is comparable to that of freely diffusible GFP [18]. It is interesting to note that the predominantly car- rier-independent cycling of unphosphorylated STAT1 was strongly affected by GFP, while the carrier- dependent nuclear import of tyrosine-phosphorylated STAT1 was not perturbed. Previous work has shown that the transport rate of protein cargo is dependent predominantly on its size and surface properties T. Meyer et al. GFP-tagging of STAT1 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS 823 [24–26]. Unphosphorylated and phosphorylated STAT1 probably are very similar in terms of their molecular mass, as there is evidence that both exist as stable dimers (molecular mass  170 kDa) [27]. Unphosphorylated STAT1 enters the nucleus without the assistance of karyopherins via direct interactions with the nuclear pore [9,28]. Two regions at the STAT1 C-terminus have been implicated in the carrier- independent nuclear translocation, the linker domain and the C-terminal transactivation domain [9,21]. In addition, the serine phosphorylation of the transactiva- tion domain was shown to enhance nuclear export of STAT1 [21]. However, as STAT1-GFP is highly ser- ine-phosphorylated (not shown), it is likely that the presence of the GFP protein interferes with the bind- ing of STAT1 to the nuclear pore proteins, thus redu- cing its ability to engage in productive interactions. Phosphorylated STAT1 dimers, on the other hand, cannot traverse the nuclear pore on their own, but require both importin a and importin b in order to enter the nucleus [9,12,13,23]. The karyopherins may function as chaperones during translocation through the pores. Irrespective of the presence of the GFP domain, these large proteins (molecular mass > 58 kDa) are likely to provide extensive interfaces with the nuclear pore that are necessary for the pas- sage of activated STAT1. It is well known that genetically encoded fluorescent tags can perturb the behavior of the acceptor protein. This study demonstrates that predominantly carrier- independent translocation mechanisms are altered by the addition of a GFP-fluorescent tag, while carrier- dependent import was not affected. An increasing number of signaling proteins is known to use both car- rier-dependent as well as carrier-independent mecha- nisms during nucleocytoplasmic cycling [29]. In those cases the mechanistically distinct translocation events have to be analyzed separately and both without and with GFP-tagging, in order to identify the possible limitations of GFP-tagged derivatives. Experimental procedures Plasmids Mammalian expression plasmids encoding wild-type human STAT1 and STAT1 cDNA fused carboxy-terminally to green fluorescent protein were described [10,30]. The construct pSTAT1-NES-GFP was generated by PCR ampli- fication of pGST-NES-GFP using the primer pair 5¢-ATA TATGGATCCAGATAAAGATGTGAATGAG-3¢ and 5¢-CGCCCCGACACCCGCCAACACCC-3¢ and Vent DNA polymerase (New England Biolabs, Frankfurt am Main, Germany). The plasmid pGST-NES-GFP encodes residues 367–427 of human STAT1, which confers NES activity [28]. The PCR product was subsequently digested with BamHI and NotI and inserted into the corresponding sites of pSTAT1-GFP. The ATG start codon of GFP was mutated to TAG to prevent expression of the GFP domain, thus generating pSTAT1-NES. Site-directed mutagenesis was done with the Quik-Change kit (Stratagene, Amster- dam, the Netherlands) and specific primers. Recombinant STAT1 proteins were purified from Sf9 insect cells that had been infected with baculovirus transfer vectors (pFastBac) encoding wild-type STAT1 or STAT1-GFP [9]. Cell culture and DNA transfections Human HeLa cells, 393T cells, and U3A cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 10% fetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin ⁄ streptomycin in a humidified 7% CO 2 atmosphere. For immunocytochemical studies the cells were passaged on poly l-lysine-coated glass coverslips in 12-well plates. The next day the cells were transiently transfected with 0.8 lgÆwell )1 expression plasmid by the Lipofectamine method according to the manufacturer’s instructions (Invitrogen, Karlsruhe, Germany). Twenty-four hours post-transfection the cells were either stimulated with 5ngÆmL )1 IFNc (Biomol, Hamburg, Germany) for the indicated times or left unstimulated. For CRM1 inhibition the cells were exposed to 10 ngÆmL )1 leptomycin B (Sigma, Munich, Germany). Purification of recombinant proteins STAT1 and STAT1-GFP, both of which contained a C-ter- minal Strep-tag, were expressed in baculovirus-infected Sf9 cells and purified from cell lysates using a Strep-Tactin col- umn [9]. Both STAT1 variants were concentrated by ultra- filtration in NaCl ⁄ P i . Microinjection Purified recombinant STAT1 proteins were injected at a concentration of 1 mgÆmL )1 into either the cytosol or nuc- leus of HeLa cells. As a marker for the injection site, 0.2 mgÆmL )1 BSA coupled to fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate (both from Sigma) was present in the injection solution. Microinjections were performed with plastic capillaries (Femtotips; Eppendorf, Hamburg, Germany) and the Transjector 5246 attached to the Micromanipulator 5171 (Eppendorf). Injection of GFP- expressing cells was monitored under an inverted micro- scope (Axiovert 25) equipped with UV light emission (Zeiss, Oberkochen, Germany). Typically, 30 cells were injected within 10 min using a maximal pressure of 40 hPa. After GFP-tagging of STAT1 T. Meyer et al. 824 FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... were normalized by the addition of U3A whole cell extracts, such that identical amounts of activated STAT1 were incubated with the radioactive probe For supershift assays the STAT1- specific antibody C-24 or an anti-GFP polyclonal antibody (obtained from rabbits immunized with purified bacterially expressed GFP-GST fusion protein) was used at a concentration of 40 lgÆmL)1 Acknowledgements The authors thank... and nuclear accumulation of the transcription factor Stat1 Genes Dev 17, 1992–2005 Sekimoto T, Nakajima K, Tachibana T, Hirano T & Yoneda Y (1996) Interferon-c-dependent nuclear import of Stat1 is mediated by the GTPase activity of Ran ⁄ TC4 J Biol Chem 271, 31017–31020 Sekimoto T, Imamoto N, Nakajima K, Hirano T & Yoneda Y (1997) Extracellular signal-dependent nuclear import of Stat1 is mediated by nuclear... Jr (1996) The rapid inactivation of the nuclear tyrosine phosphorylated Stat1 depends upon a protein tyrosine phosphatase EMBO J 15, 6262–6268 Axelrod D, Koppel DE, Schlessinger J, Elson E & Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics Biophys J 16, 1055–1069 Patterson GH, Knobel SM, Sharif WD, Kain SR & Piston DW (1997) Use of the green fluorescent. .. Constitutive and IFN-c-induced nuclear import of STAT1 proceed through independent pathways EMBO J 21, 344–354 ´ Xu L & Massague J (2004) Nucleocytoplasmic shuttling of signal transducers Nat Rev Mol Cell Biol 5, 209–219 Begitt A, Meyer T, van Rossum M & Vinkemeier U (2000) Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain Proc Natl Acad Sci... phosphorylation of a latent cytoplasmic transcription factor Science 257, 809–813 Meyer T & Vinkemeier U (2004) Nucleocytoplamic shuttling of STAT transcription factors Eur J Biochem 271, 4606–4612 Marg A, Shan Y, Meyer T, Meissner T, Brandenburg M & Vinkemeier U (2004) Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1dependent nuclear export control the subcellular distribution of latent Stat1. .. Horinouchi S & Yoshida M (1998) Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1 Exp Cell Res 242, 540–547 McBride KM, Banninger G, McDonald C & Reich NC (2002) Regulated nuclear import of the STAT1 transcription factor by direct binding of importin-a EMBO J 21, 1754–1763 Feldherr CM & Akin D (1990) The permeability of the nuclear envelope in dividing and nondividing cell... tyrosine-phosphorylated STAT1 GFP-tagging of STAT1 (Cell Signaling, Frankfurt am Main, Germany), which was diluted 1 : 1000 in TBS-T Then the membranes were washed three times in TBS-T and relevant bands were detected by incubating with horseradish peroxidase-conjugated secondary antibody (Dako) using the chemiluminescence reaction kit (Amersham Biosciences, Freiburg, Germany) The membranes were stripped off bound... Koster M & Hauser H (1999) Dynamic redistribution ¨ of STAT1 protein in IFN signaling visualized by 826 18 19 20 21 22 23 24 25 26 27 28 29 30 GFP fusion proteins Eur J Biochem 260, 137–144 Lillemeier BF, Koster M & Kerr IM (2001) STAT1 ¨ from the cell membrane to the DNA EMBO J 20, 2508–2517 Koster M, Frahm T & Hauser H (2005) Nucleocytoplas¨ mic shuttling revealed by FRAP and FLIP technologies Curr... single phosphotyrosine residue of STAT91 required for gene activation by interferon-gamma Science 261, 1744–1746 5 Shuai K, Horvath CM, Huang LH, Qureshi SA, Cowburn D & Darnell JE Jr (1994) Interferon activation of the transcription factor Stat91 involves dimerization FEBS Journal 274 (2007) 815–826 ª 2007 The Authors Journal compilation ª 2007 FEBS 825 GFP-tagging of STAT1 6 7 8 9 10 11 12 13 14 15...T Meyer et al the indicated incubation periods at 37 °C the cells were fixed for 10 min at )20 °C in cold methanol Injected STAT1 was detected with monoclonal antibody C-136 as described [9] Immunocytochemistry Indirect immunofluorescence microscopy of untransfected cells and cells transiently expressing recombinant STAT1 was done with affinity-purified rabbit polyclonal STAT1specific antibody C-24 . the inclusion of the export signal, the expression of the GFP domain no longer reduced the transcriptional activity of STAT1 (Fig. 6C). In the presence of LMB, which specifically inactivates the exportin. GFP-tagging impairs the nucleocytoplasmic translocation specifically of unphosphorylated STAT1 To directly analyze the influence of GFP-tagging on the nucleocytoplasmic translocation of STAT1, we per- formed. gels. The positions of homodimers of endogenous STAT1 (lower mark), of homodimers of recombinant STAT1- GFP (upper mark), and of heterodimers thereof (asterisk) are indicated at the right margin of