STAT1 from the cell membrane to the DNA
2001; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.1093/emboj/20.10.2508
ISSN1460-2075
Autores Tópico(s)RNA Research and Splicing
ResumoArticle15 May 2001free access STAT1 from the cell membrane to the DNA Björn F. Lillemeier Björn F. Lillemeier Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Mario Köster Mario Köster Gene Regulation and Differentiation, GBF – National Research Institute for Biotechnology, Braunschweig, Germany Search for more papers by this author Ian M. Kerr Corresponding Author Ian M. Kerr Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Björn F. Lillemeier Björn F. Lillemeier Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Mario Köster Mario Köster Gene Regulation and Differentiation, GBF – National Research Institute for Biotechnology, Braunschweig, Germany Search for more papers by this author Ian M. Kerr Corresponding Author Ian M. Kerr Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Author Information Björn F. Lillemeier1, Mario Köster2 and Ian M. Kerr 1 1Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 2Gene Regulation and Differentiation, GBF – National Research Institute for Biotechnology, Braunschweig, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2508-2517https://doi.org/10.1093/emboj/20.10.2508 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The binding of interferons (IFNs) to their receptors leads to the phosphorylation and activation of signal transducers and activators of transcription (STATs), and their translocation from the cytoplasm to the nucleus. The mechanisms by which the STATs move to the nuclear pore are not, however, known. Here it is shown that IFN-α and -γ signalling and STAT1 translocation are independent of the actin cytoskeleton or microtubules. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) experiments, the mobility of a fusion protein of STAT1 with green fluorescent protein (STAT1–GFP) was compared with that of GFP and protein kinase C–GFP. In IFN-γ-treated and control cells, cytoplasmic STAT1–GFP shows high, energy-independent, mobility comparable to that of freely diffusible GFP. A random walk model for movement of STAT1 from the plasma membrane to the nuclear pore is, therefore, indicated. Nuclear STAT1–GFP showed similar high mobility, with exclusion from nucleoli, consistent with high rates of association and dissociation of STAT1–DNA and/or STAT1–protein complexes in the nucleoplasm of the cell. Introduction Appropriate localization of proteins is crucial for their physiological function and regulation. Proteins transmit incoming signals by interaction with other molecules with or without movement into another location in the cell. Activation of transcription factors can occur in the cytoplasm or at the cell membrane. On activation, they translocate to the nucleus through nuclear pores (Kaffman and O'Shea, 1999). Little is known, however, about the mechanisms of transport to the nuclear pores. Movement through the cytoplasm could, a priori, be active along microtubules as observed for p53 (Giannakakou et al., 2000), or passive, by diffusion, as in a random walk. The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathways, first identified in the interferon (IFN) systems (Darnell et al., 1994), are activated by a wide range of cytokines and growth factors. There are four known mammalian members of the JAK family of protein tyrosine kinases: JAK1, JAK2, JAK3 and Tyk2 (Wilks, 1989; Firmbach-Kraft et al., 1990; Harpur et al., 1992; Silvennoinen et al., 1993; Johnston et al., 1994; Witthuhn et al., 1994) and seven mammalian STAT genes (STATs 1–6, including STATs 5A and B; Fu, 1992; Fu et al., 1992; Schindler et al., 1992a; Akira et al., 1994; Hou et al., 1994; Wakao et al., 1994; Yamamoto et al., 1994; Zhong et al., 1994; Quelle et al., 1995). Upon ligand binding, the receptor-associated JAKs become activated by auto- and transphosphorylation, and phosphorylate the receptor. STATs are recruited to the JAK–receptor complexes, phosphorylated, released and migrate to the nucleus to activate transcription (Ihle et al., 1995; Schindler and Darnell, 1995; Duhe and Farrar, 1998; Leonard and O'Shea, 1998; Stark et al., 1998; Yeh and Pellegrini, 1999). JAK1 and Tyk2, and JAK1 and JAK2 are associated with the IFN-α/β and IFN-γ receptors, respectively (Muller et al., 1993a; Watling et al., 1993). STATs 1–5 can be activated in response to IFN-α. Here, however, we will be largely concerned with the activation of STAT1, the only STAT activated in response to IFN-γ in the human cell systems used. Retention of non-activated STAT1 in the cytoplasm does not reflect anchoring or inhibition of shuttling (McBride et al., 2000). On release from the JAK–receptor complex, activated STAT1 dimers migrate to the nuclear pore. No 'classical' nuclear localization sequence (NLS) has been detected in STAT1. However, interaction of activated STAT1 with importin NPI-1 initiates translocation through the nuclear pore (Sekimoto et al., 1997). The latter takes time and is energy dependent. Consequent upon these requirements, the nuclear membrane forms an effective barrier to the translocation of non-activated STAT1 (Köster and Hauser, 1999; Figure 3). Dephosphorylation of STAT1 in the nucleus (Haspel and Darnell, 1999; McBride et al., 2000) and export to the cytoplasm (McBride et al., 2000; Mowen and David, 2000) control recycling of STAT1 and maintain responsiveness of the cell. Here we have investigated the dependence of IFN-α and -γ signalling on the cytoskeleton. Also, using a known biologically active STAT1–GFP (where GFP is green fluorescent protein) (Köster and Hauser, 1999; Figure 2), we have examined for IFN-γ how STAT1 moves from the plasma membrane JAK–receptor complex to the nuclear pore, and the nuclear mobility of STAT1. The data are consistent with random walk models for the movement of activated STAT1 in both compartments. Results Interferon signalling is not dependent on an intact cytoskeleton The inhibitors cytochalasin D, which prevents actin polymerization, and nocodazole, which disrupts the formation of microtubules, were used to determine the influence of these structural elements on IFN signalling. 2fTGH fibrosarcoma cells were pre-incubated in the absence or presence of either drug for 90 min, then stimulated with IFN-α or -γ for 15 h under continued drug treatment. The efficacy of the drugs was confirmed by changes in cell morphology and the loss of cell adherence. Transcriptional activity upon IFN stimulation was measured by RNase protection assays using protection probes for the typical IFN-stimulated genes (ISGs) p48, 6-16, IRF1 and 9-27 (Figure 1A). The data were quantified by phosphorimaging corrected for actin levels (Figure 1B). The transcriptional response to IFN-α or -γ was not affected by either drug treatment in comparison with untreated controls. In additional experiments, neither the kinetics nor the dose responses were significantly affected (data not presented). Accepting this lack of dependence on the cytoskeleton, the mobility of STAT1 was further investigated in live cells using a known functional STAT1–GFP (Köster and Hauser, 1999). Figure 1.Interferon signalling is not dependent on an intact cytoskeleton. (A) 2fTGH cells were incubated for 90 min with cytochalasin D or nocodazole, as indicated, then treated with 103 IU/ml IFN-α or -γ for 15 h under continued drug treatment. Expression of inducible mRNAs (p48, 6-16, IRF-1, 9-27) was monitored by RNase protection (Materials and methods). (B) Quantitation of the data in (A) by PhosphorImager analysis. Fold induction was calculated after correction for the γ-actin loading control. Download figure Download PowerPoint Figure 2.STAT1–GFP is a functional transcription factor. (A and B) 2C4 (left lanes) and 2C4/STAT1–GFP cells (right lanes) were stimulated with 103 IU/ml IFN-α or -γ for 20 min. (A) Phosphorylation of STAT1–GFP. Western blot analysis of total cell lysates with an anti-P-Tyr701 specific antibody (upper panel), an anti-STAT1 antibody as loading control (middle panel) and an anti–GFP antibody to monitor for any STAT1–GFP cleavage (lower panel). No evidence for free GFP was obtained even on prolonged exposure (data not shown). (B) DNA binding of STAT1–GFP was analysed by EMSA of whole-cell extracts with an SIE probe. (C) The expression of STAT1 and STAT1–GFP in the 2C4/STAT1–GFP population was compared by analysing a series of dilutions of a total cell lysate by western blot analysis with antibody to STAT1 (upper panel). The relative fluorescence intensity of 54 single cells was analysed by confocal microscopy (left ordinate, lower panel; Materials and methods). The ratio of STAT1–GFP to endogenous STAT1 (right ordinate, lower panel) was calculated from the two data sets. The dotted line shows both the average fluorescence (left scale) and the average expression relative to STAT1 (right scale) of STAT1–GFP in the population. Download figure Download PowerPoint Figure 3.FLIP analysis. (A) Cytoplasmic and (B) nuclear fluorescence. (A) 2C4 cells stably transfected with STAT1–GFP without (row 1) and after 15 min of IFN-γ treatment (103 IU/ml; row 2), GFP (row 3) and PKC–GFP without (row 4) and after 5 min of TPA treatment (Materials and methods; row 5). Every image in a row contains the same cells. The bleach region in the cytoplasm is indicated with a white square and fluorescence intensity is shown in false colour code (vertical bar, top right). Each row shows the fluorescence prior to bleaching (0 s) and after three consecutive 90 s bleach periods (90, 180 and 270 s). In the case of PKC–GFP, after TPA the plane of focus was the membrane parallel to the coverslip (row 5). (B) 2C4 cells stably transfected with STAT1–GFP and treated with IFN-γ at 103 IU/ml for 25 min (row 1) and GFPnls (row 2). Each row shows the fluorescence prior to bleaching and after three consecutive 30 s bleach periods (0, 30, 60 and 90 s, respectively) of the nuclear area bounded by the white square. Download figure Download PowerPoint STAT1–GFP is a functional transcription factor The construction and characterization of a functional STAT1–GFP, the behaviour of which is indistinguishable from native STAT1 with respect to its activation and translocation in and out of the nucleus, have already been described (Köster and Hauser, 1999). Here, the comparable restoration of IFN responses to STAT1-negative U3A cells by STAT1 and STAT1–GFP was confirmed by RNase protection assays monitoring the induction of representative sets of IFN-γ- and IFN-α-inducible genes (Köster and Hauser, 1999 and data not shown). For the remainder of the experiments, wild-type 2C4 cells were used in preference to the mutiply mutagenized U3A cells. The function of stably transfected STAT1–GFP in 2C4 cells again appeared indistinguishable from that of the, in this case, endogenous STAT1. The STAT1– GFP is comparably tyrosine phosphorylated/activated (Figure 2A), shows comparable DNA-binding activity (Figure 2B) and is efficiently translocated to the nucleus in response to IFN-γ stimulation (Figure 5; see Köster and Hauser, 1999). Comparison of endogenous STAT1 levels with those of the stably transfected STAT1–GFP by western blotting shows an average 3-fold overexpression of STAT1–GFP in the population (Figure 2C, top). The fluorescence of single cells (Figure 2C, bottom), together with the data from the western blot analyses, indicate that the majority of single cells express STAT1–GFP to levels comparable with or up to 10-fold higher than endogenous STAT1. No significant difference was observed between high- and low-expressing cells in the single-cell-based fluorescence studies below. Figure 4.FRAP analysis. (A) Cytoplasmic and (B) nuclear fluoresence. (A) 2C4 cells stably transfected with STAT1–GFP without (row 1) and after 15 min of IFN-γ treatment at 103 IU/ml (row 2), GFP (row 3) and PKC–GFP without (row 4) and after 5 min of TPA treatment (Materials and methods, row 5). Cells were bleached (Materials and methods) for 17.5 s in an area comparable to the bleach area in the FLIP analysis (white squares, Figure 3). The recovery of fluorescence in the bleach area (squares) and four surrounding areas (triangles) of the same size (averaged) was quantified over a period of further 60 s in 1 s intervals. The fluorescence was normalized to the remaining fluorescence in the cell at the end of the experiment. The right-hand panels show data for cells incubated for 20 min with sodium azide and 2-deoxyglucose to deplete ATP, prior to bleaching. Each graph represents the average of data from 10 single cells. (B) FRAP analysis of the nuclear fluorescence of 2C4 cells stably transfected with STAT1–GFP and treated with 103 IU/ml IFNγ for 25 min (row 1) and GFPnls (row 2). Bleaching, recovery, ATP deprivation (right hand panels) and symbols are as in (A). The apparently more extensive initial bleach level for membrane-associated PKC–GFP (row 5, squares) compared with that in all other rows reflects the slower movement of the membrane-associated PKC–GFP than the freely diffusing GFP and other GFP constructs during the switch of the laser from the bleach to the record modes. Download figure Download PowerPoint Figure 5.Nuclear translocation of STAT1–GFP is not dependent on active JAK–receptor complexes. (A) Inhibition of JAK-dependent STAT1 activation by staurosporine. 2C4 cells were pre-treated with 100 or 500 nM staurosporine for 2, 5, 15 and 30 min, and stimulated with IFN-γ at 103 IU/ml for a further 20 min in the continued presence of the drug. Activation of STAT1 was monitored by EMSA of whole-cell extracts with an SIE probe in comparison with extracts from cells without drug treatment. [Similar results were obtained when the phosphorylation of STAT1 was monitored directly by (less sensitive) western blot analysis.] (B) Nuclear translocation of pre-activated STAT1. 2C4/STAT1–GFP cells were incubated with 103 IU/ml IFN-γ for 15 min to activate the STAT1–GFP. Staurosporine (500 nM) was added to half of the cells and both the staurosporine-treated and non-staurosporine-treated cells incubated for a further 5 and 15 min (to yield the 20 and 30 min time points, respectively). At 0, 15, 20 and 30 min, samples were fixed in paraformaldehyde and the distribution of STAT1–GFP analysed by confocal imaging. Fluorescence intensities are shown in false colour code (vertical bar, top right). Download figure Download PowerPoint High mobility of cytoplasmic and nuclear STAT1–GFP: FLIP and FRAP analyses Little is known about the mobility of STAT1 in the cytoplasm or the nucleus. In order to address these issues, fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) analyses were carried out to compare the mobilities of stably expressed STAT1–GFP, GFP and protein kinase C (PKC)–GFP in the cytoplasm and nucleus of live 2C4 cells before and after treatment with IFN-γ or phorbol ester. FLIP analysis of cytoplasmic STAT1–GFP. In FLIP analysis, a small region of the cytoplasm (white box, Figure 3; the intensity of the fluorescence signal is indicated by a 'false colour' bar to the right of the images) was bleached by scanning for three consecutive periods of 90 s with maximum laser intensity and the fluoresence of the whole cell monitored. To make sure there was no generalized bleaching effect due to the imaging, every bleached cell had an unbleached neighbouring cell in the same image, which maintained high fluorescence (Figure 3). The behaviour of non-activated and IFN-γ-activated STAT1 was compared with GFP, known to diffuse freely in cells, and free and membrane-bound (more slowly diffusing) PKC–GFP. Cells expressing GFP (Figure 3A, third row), STAT1–GFP, before and after treatment with IFN-γ (Figure 3A, rows 1 and 2) and free PKC–GFP without phorbol ester treatment (Figure 3A, row 4) lost most of their cytoplasmic fluorescence after the first 90 s of bleaching. Further bleaching to totals of 180 and 270 s resulted in the entire loss of cytoplasmic fluorescence. For STAT1–GFP and PKC–GFP, the nuclear envelope imposes a barrier to free diffusion. In these short time periods, no STAT1–GFP is released from the nucleus (Figure 3A, rows 1 and 2). [There is a low level of constitutively activated STAT1–GFP in the nucleus of the control cells (Figure 3A, row 1).] Conversely, PKC–GFP does not enter the nucleus of control cells (Figure 3A, row 4). In cells treated with phorbol ester, PKC–GFP is activated and translocated to the membrane. The reduced mobility of membrane-associated PKC–GFP, visualized by focusing on the lower membrane, was obvious: even after three periods of bleaching, only the boxed area and the close surroundings showed a loss of fluorescence in these cells (Figure 3A, row 5). Depletion of ATP (see below) had no influence on the results of this type of FLIP analysis. The data indicate that most of the STAT1–GFP molecules passed through the region being bleached within 270 s, moving rapidly throughout the cytoplasm in a random ATP-independent fashion. This is in contrast to the data for the more slowly diffusing, membrane-associated, activated PKC–GFP. FRAP analysis of cytoplasmic STAT1–GFP. In order to have a more quantitative measurement of mobility to permit the calculation of the amount of any immobile fraction of STAT1–GFP, FRAP analyses were performed on the same stably transfected cell lines as were used for the FLIP analysis A region in the cytoplasm was bleached for a period of 17.5 s and the effect on the fluorescence intensity in this area was measured for a further 60 s. In parallel, regions surrounding the bleached region were measured for the same period of time. To investigate whether the movement of the fusion proteins was an energy-dependent process, a second set of cells was depleted of ATP by pre-treatment with sodium azide and 2-deoxyglucose prior to analyses (right-hand panels, Figure 4A). This treatment is sufficient to inhibit ATP-dependent fluid-phase endocytosis of fluorescent Cy3-labelled antibodies (Wubbolts et al., 1996 and data not shown). With or without depletion of ATP, the recovery rates in the bleached regions of the cytoplasm (empty squares, Figure 4A) for GFP (Figure 4A, third panels down), STAT1–GFP before and after IFN-γ treatment (Figure 4A, top and second panels), and PKC–GFP without phorbol ester treatment (Figure 4A, fourth panels down) were similar. In contrast, the recovery of PKC–GFP after phorbol ester treatment was slower and never complete. Again, this was not influenced by the depletion of ATP (Figure 4A, bottom panels). Comparison of the bleached region (empty squares) versus the surrounding regions (filled triangles) did not show, for GFP, STAT1–GFP before and after IFN-γ treatment, and PKC–GFP without phorbol ester, a significant difference such as would have resulted from the presence of an immobile fraction, as is the case for PKC–GFP after phorbol ester (contrast rows 1–4 with row 5, Figure 4A). As little as 1% of immobile GFP would have been detected in this type of analysis. In contrast to GFP per se (Figure 4A, row 3), very small differences directly after the bleaching were detectable in the cells expressing STAT1–GFP and PKC–GFP (Figure 4A, rows 1, 2 and 4). These most likely reflect differences in size and hence in mobility of the fusion proteins versus free GFP. The comparison of the bleached region to the surrounding regions for PKC–GFP after phorbol ester treatment showed an immobile fraction of ∼5 ± 2% (n = 10) for the membrane-associated PKC– GFP (Figure 4A, bottom row). Nuclear translocation of pre-activated STAT1 does not depend on continued activity of JAK–receptor complexes At any given instant in time, a small percentage (below the 1% that would be detected by FRAP) of the STAT1–GFP might be associated with a 'hard-wired' directional transport mechanism linking active JAK–receptor complexes to nuclear pores. In the presence of the kinase inhibitor staurosporine (Haspel and Darnell, 1999) and hence the absence of continued JAK–receptor activity, pre-activated STAT1–GFP is efficiently translocated into the nucleus. In initial electrophoretic mobility shift assay (EMSA) analyses in 2C4 cells, adding staurosporine before stimulation with IFN-γ showed that the drug is effective in inhibiting JAK activation of STAT1 in 5-fold wild type) and low (≈ wild type) expressing cells. The results do not, therefore, reflect overexpression artefacts. The FLIP and FRAP experiments provide insight into the nature of STAT1 movement. STAT1–GFP mobility, with or without ligand stimulation, is comparable to that of freely diffusible GFP (Figures 3 and 4). It is rapid, with all of the STAT1 molecules moving through all locations in the cytoplasm or nucleus within minutes, and independent of ATP. An immobile fraction of STAT1–GFP was not detected and can be excluded down to a level of ∼1% of the total STAT1–GFP (Figure 4). A priori, at any given instant in time, a small percentage of the STAT1–GFP might be associated with a 'hard-wired' directional transport mechanism linking the JAK–receptor complex to the nuclear pore. However, in the presence of staurosporine and hence the absence of an enzymatically active JAK–receptor complex, a substantial fraction (∼50%) of total STAT1–GFP is translocated into the nucleus (Figure 5). Thus, pre-activated, randomly distributed STAT1–GFP is translocated to the nucleus as efficiently as in non-staurosporine-treated cells (Figure 5). One still cannot exclude the possibility that, at any given time, a small percentage of STAT1–GFP (too low to be detected by FLIP or FRAP) is transported directionally to the nuclear pore. Access to any such putative transport system would, however, have to be available to randomly distributed pre-activated STAT1–GFP. In this model, the putative translocation system would conceptually be an extension of the nuclear pore–importin complex, with random access through free diffusion after release from the receptor. In an alternative approach, STAT1 generated from a completely foreign receptor can sustain an IFN-γ-like response. This favours modular signalling and 'soft' rather than 'hard' wiring of the IFN-γ-induced cytoplasmic signal transduction pathway(s) (V.Arulampalam, B.Strobl, H.Is'harc and I.M.Kerr, in preparation). Overall, the data are consistent with free diffusion of the STAT1 from the membrane JAK–receptor complex to the nuclear pore. Activated STAT1 forms a dimer, dimerization being required for recognition by importin NPI-1 and translocation through the nuclear pore (Sekimoto et al., 1997). Larger STAT1 complexes have been reported (Lackmann et al., 1998; Ndubuisi et al., 1999), and highly dynamic interactions with randomly distributed cytoplasmic complexes remain perfectly possible. Such interactions would not, however, confer directionality to the movement of STAT1. The behaviour of STAT1–GFP in the nucleus is also of interest. Nucleoplasmic movement is again rapid, random, independent of ATP and comparable to that of freely diffusible GFP (Figures 3B and 4B). STAT1–GFP, in contrast to free GFP, is, however, excluded from nucleoli (Figures 3 and 6), presumably through interaction with nucleoplasmic protein–DNA complexes. Such interactions would have to be highly dynamic, with high rates of association and dissociation, to sustain both the high mobility and localization of the STAT1–GFP. Interactions of this type with similar effects on localization have recently been described for a number of nuclear proteins (Phair and Misteli, 2000). More particularly, for the glucocorticoid receptor, association with transcriptional complexes is similarly highly dynamic, in accord with a 'hit and run' model rather than the formation of stable initiation complexes (McNally et al., 2000). As the loss of specific G
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