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First published online 28 August 2007
doi: 10.1242/jcs.03482

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Nucleocytoplasmic shuttling of persistently activated STAT3

Andreas Herrmann^1,*,, Michael Vogt^1,*, Martin Mönnigmann^2,, Thomas Clahsen¹, Ulrike Sommer¹, Serge Haan¹, Valeria Poli³, Peter C. Heinrich¹ and Gerhard Müller-Newen^1,¶

¹ Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany
² Lehrstuhl für Prozesstechnik, RWTH Aachen, Templergraben 55, 52056 Aachen, Germany
³ Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, University of Turin, Via Nizza 52, 10126 Torino, Italy

View larger version (36K): [in this window] [in a new window]   Fig. 1. v-Src expression leads to constitutive activation of wild-type and fluorescent STAT3 fusion proteins. (A) COS-7 cells were transiently transfected with v-Src and STAT3 as indicated and stimulated with 20 ng/ml IL-6 and 1 µg/ml sIL-6R for 30 minutes or left untreated. Whole cell lysates were subjected to SDS-PAGE and western blotting. STAT3 tyrosine phosphorylation was detected with a pY705-STAT3 antibody and as a loading control STAT3 was counterstained with a specific STAT3 antibody. (B) Transiently expressed STAT3-YFP and mutant fusion proteins as indicated were precipitated from COS-7 cell lysates with a GFP antibody that recognizes YFP and CFP. Western blot analyses of precipitates were performed for pY705-STAT3 and counterstained for total STAT3 and YFP. hc, heavy chain of the precipitating antibody. (C) Nuclear extracts were prepared from MEFfl/fl, MEF/ or MEF/STAT3-YFP cells stimulated with 20 ng/ml IL-6 and 1 µg/ml sIL-6R for 30 minutes as indicated. STAT3, STAT1 and STAT3-YFP DNA-binding to a [32P]-labelled SIE-probe was detected by EMSA. Antibodies against STAT3 and GFP were used for the supershift experiment. (D) MEF/STAT3-YFP cells were co-transfected with v-Src, fixed and stained for pY416-Src. Fixed cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. A Western blot of lysates of MEF/STAT3-YFP cells was analyzed for pY705-STAT3 and counter-stained for total STAT3. (E) MEF/STAT3-CY cells were treated and analyzed as described in D. Bars, 10 µm. A Western blot of lysates of MEF/STAT3-CY cells was analyzed for pY705-STAT3 and counter-stained for total STAT3. (F) MEFfl/fl and MEF/STAT3-YFP cells were transfected with v-Src, Fyn, Abl, or BCR-Abl or left untransfected for IL-6 stimulation and the negative control as indicated. Whole cell lysates were subjected to western blotting and analyzed for pY705-STAT3 and total STAT3 as a loading control.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Jak1, SOCS3 and PIAS3 in v-Src-mediated STAT3 activation. (A) Parental and Jak1-deficient human fibrosarcoma cells were transiently transfected with STAT3-YFP. Cells were stimulated with 20 ng/ml IL-6 and 1 µg/ml sIL-6R for 30 minutes (middle panel) or left unstimulated (upper panel). Cells transiently co-transfected with v-Src were left unstimulated (lower panel). Cells were fixed with paraformaldehyde and, as a nuclear marker, lamin A/C was stained. v-Src was stained with an antibody recognizing pY416-Src. Cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. (B) HepG2 human hepatocellular carcinoma cells were transiently transfected with v-Src or with an empty vector. A STAT3-responsive luciferase reporter gene construct under control of the 2M-promoter was co-transfected. Cells were incubated with 20 ng/ml IL-6 or left unstimulated for 24 hours in the presence or absence of 100 ng/ml Jak inhibitor 1 as indicated. Reporter gene assays were performed in triplicate and standard deviations were calculated. (C) HepG2 cells were transiently transfected with YFP-SOCS3 and STAT3-CFP (upper and middle panel) or with YFP-SOCS3, STAT3-CFP and v-Src (lower panel). Stimulation was performed with 20 ng/ml IL-6 for 30 minutes. Fixed cells were stained for lamin A/C or pY416-Src, respectively. Images were taken by confocal laser-scanning microscopy. Bars, 10 µm. (D) HepG2 cells were transiently transfected with SOCS3, v-Src and a 2M reporter gene construct as indicated. Reporter gene assays of IL-6 stimulated (+) and unstimulated (–) cells were performed as described in B. (E) HepG2 cells were transiently transfected with FLAG-PIAS3 and STAT3-CFP (upper and middle panel) or with FLAG-PIAS3, STAT3-CFP and v-Src (lower panel). Stimulation was performed with 20 ng/ml IL-6 for 30 minutes. Fixed cells were stained for FLAG-tagged PIAS3 using a FLAG antibody and an antibody recognizing pY416-Src, respectively. Cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. (F) HepG2 cells were transiently transfected with v-Src, FLAG-PIAS3 and a 2M reporter gene construct as indicated. Reporter gene assays were performed as described in D.

View larger version (63K): [in this window] [in a new window]   Fig. 3. iFLAP imaging. (A) Schematic representation of the iFLAP imaging approach. (Left) Using the 514 nm laser of the confocal laser-scanning microscope the YFP part of STAT3-CY can be selectively bleached. (Right) The images of STAT-CY in the YFP and CFP channels of the confocal microscope (original data) are processed into zero signal intensity depicted as blue (intensity= 1–(iYFP/iCFP). Selective bleaching of YFP within the rectangular bleaching-ROI (ROI=region of interest) generates a subpopulation of STAT3-CY molecules that are only detected in the CFP-channel. These molecules generate a signal in the processed data channel. They diffuse freely through the cytoplasm and finally appear within the nucleus. (B) HepG2 transiently transfected with STAT3-CY were analyzed in a perfusion chamber for live cell imaging at the confocal laser-scanning microscope. Two neighbouring cells were selected for imaging (upper-left image shows the merged YFP and CFP channels). The YFP and CFP fluorescence along the red line are shown in the upper-right diagram as yellow and blue graphs, respectively (N, fluorescence intensity within the nucleus). The lower cell was selected for the iFLAP experiment. A bleaching-ROI (white rectangle) was placed into its cytoplasmic compartment, the bleaching procedure was performed, and fluorescence intensity signals of both CFP and YFP were recorded every 5 minutes. Detected signals were recalculated by using the algorithm described in A to generate a signal that represents the distribution of the bleached subpopulation of STAT3-CY over time. As a control the second cell was monitored without bleaching. Relative fluorescence is depicted in false colour mode corresponding to its intensities. Fluorescence intensity profiling (red arrows and right diagrams) is depicted to show the increasing signal in the nuclear compartment. (C) The same experiment was performed as described in B with HepG2 cells coexpressing STAT3-CY and v-Src. (D) The same experiment was performed as described in C in the presence of 100 ng/ml ratjadone A.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Quantitative iFLAP. (A) HepG2 cells co-transfected with STAT3-CY and v-Src were analyzed in a perfusion chamber for live cell imaging at the confocal microscope. CFP and YFP fluorescence intensities were monitored exclusively in regions of interest (ROI, numbered red circles in the images) over time. The initial cytoplasmic bleaching was performed in the bleach ROI (white circle) over the time indicated by the red bar on the x-axis in the diagram. The diagram represents the mean CFP (blue lines) and YFP fluorescence intensities (orange lines) in the three ROIs over time. The red numbers in the diagrams correspond to the cytoplasmic (1), nuclear (2) and background (3) detection ROIs. A representative experiment is shown. (B) iFLAP experiments were performed with HepG2 cells transiently transfected with STAT3-CY and with stably transfected MEF/-STAT3-CY cells co-transfected with v-Src or empty vector as described in A. The resulting curves were analyzed by mathematical modelling and computational parameter estimation. The bar chart depicts the mean values and s.d. of the exponential coefficient as a measure for the rate of shuttling.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Inhibition of nuclear export leads to a decrease in v-Src-mediated STAT3 activation and gene induction. (A) MEF/STAT3-YFP cells were stimulated with 20 ng/ml IL-6 and 0.5 µg/ml sIL-6R for the indicated times and treated with 100 ng/ml ratjadone A (lower panel) or left untreated (upper panel). Fixed cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. A western blot of corresponding lysates of MEF/STAT3-YFP cells was analyzed for pY705-STAT3 and counterstained for total STAT3. (B) MEF/STAT3-YFP cells were transfected with v-Src and treated for 2 hours with ratjadone A as indicated. Whole cell lysates were subjected to SDS-PAGE and western blotting. STAT3 tyrosine phosphorylation was detected with a pY705-STAT3 antibody. For loading control the blot was stained with a STAT3 antibody. (C) MEF/ cells and MEF/STAT3-YFP cells were transfected with v-Src and treated with ratjadone A for 24 hours. Cell lysates were subjected to SDS-PAGE and western blotting. pY705-STAT3, STAT3 and cyclin D1 were detected by immunostaining. (D) A luciferase reporter construct under control of the cyclin D1 promoter was transfected into MEF/ and MEF/STAT3-YFP cells. v-Src was co-transfected as indicated. 48 hours after transfection cells were treated with 100 ng/ml ratjadone A for 24 hours or left untreated. Reporter gene assays were performed in triplicate and s.d. was calculated.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Disrupting nucleocytoplasmic shuttling of constitutively activated STAT3 in v-Src-transformed fibroblasts results in apoptotic cell death. Mouse 3T3 fibroblasts and v-Src-transformed mouse fibroblasts were cultivated in six-well plates for 72 hours and then subjected to further analysis. Cells were either left untreated (–), were treated with ratjadone A for the last 24 hours (+) or treated with ratjadone for 24 hours and subsequently incubated for another 24 hours without ratjadone A (+/–). (A) Phase contrast images of the cells after 72 hours. (B) Cell lysates were subjected to SDS-PAGE and western blotting. pY705-STAT3 and STAT3 were detected by immunostaining. (C) Cells were harvested, stained with annexin V-FITC and analyzed by flow cytometry. The non-treated cell population is shown in grey in all histograms. The histograms of ratjadone-A-treated cells are shown as black lines.

View larger version (34K): [in this window] [in a new window]   Fig. 7. v-Src- and IL-6-mediated STAT3 activation and its inhibition. Cytokine-induced STAT3 activation (right) is transient owing to the induction of the feedback inhibitor SOCS3. Persistent activation of STAT3 by v-Src (left) requires nucleocytoplasmic shuffling. Inhibition of nuclear export blocks the reactivation cycle. See Discussion for details.

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