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First published online August 24, 2006
doi: 10.1242/10.1242/jcs.03086

Journal of Cell Science 119, 3695-3704 (2006)
Published by The Company of Biologists 2006
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Efficient nuclear export of p65-I{kappa}B{alpha} complexes requires 14-3-3 proteins

Cristina Aguilera, Vanessa Fernández-Majada, Julia Inglés-Esteve, Verónica Rodilla, Anna Bigas* and Lluís Espinosa*

Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica, Gran Via km 2.7, Hospitalet, Barcelona 08907, Spain


Figure 1
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  		Fig. 1. p65 binds to 14-3-3 proteins in response to TNF{alpha}. (a) Pull-down experiment using GST-14-3-3{eta} and lysates from untransfected HEK-293T cells incubated with TNF{alpha} at the indicated times. Upper panel shows immunoblot with anti-p65 antibody. Ponceau staining of GST proteins is shown in the lower panel. Inputs represent 1/10 of the lysates. (b) Cell lysates from HEK-293T, untreated or treated with TNF{alpha}, were precipitated with anti-p65 antibody or rabbit IgG as a control. The presence of 14-3-3 in the precipitates was determined by immunoblotting with anti-14-3-3ß antibody recognizing different 14-3-3 isoforms ({alpha}-14-3-3). Inputs represent 1/10 of the lysates. (c) Schematic overview of the p65 protein sequence indicating the putative 14-3-3 binding domains.

 

Figure 2
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  		Fig. 2. Interaction between p65 and 14-3-3 depends on phosphorylation. (a,b) Pull-down assay with GST-14-3-3{eta} and cell lysates from TNF{alpha}-treated HEK-293T cells incubated with acid phosphatase for 30 minutes (a) or from cells incubated for 2 hours with the indicated inhibitors plus 30 minutes with TNF{alpha} (b). The presence of p65 in the precipitates was determined by western blot with anti-p65 antibody. Coomassie staining of GST proteins is shown in lower panel. Inputs represent 1/10 of the lysates. (c) In vitro kinase assay to test the capacity of total cell lysates, from untreated or TNF{alpha}-treated HEK-293T cells, to phosphorylate GST-p65 peptides including the putative 14-3-3 binding domains. Upper panels show phosphorylated peptides by autoradiography. Coomassie staining of GST proteins is shown in lower panel. (d) Cell lysates from HEK-293T transfected with the full-length GFP-p65wt and the indicated mutant plasmids, treated for 60 minutes with TNF{alpha} were precipitated with the anti-p65 antibody. Western blot analysis with anti-P-14-3-3 binding motif antibody confirmed the presence of phosphorylated 14-3-3 binding domains in p65. Precipitated GFP-p65 protein levels are shown in the lower panel. (e) Pull-down experiment using GST-14-3-3{eta} and lysates from HEK-293T cells transfected with the indicated p65 mutants and treated for 30 minutes with TNF{alpha}. Upper panel shows immunoblot with anti-p65. Coomassie staining of GST proteins is shown in lower panel. Inputs represent 1/10 of the lysates.

 

Figure 3
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  		Fig 3. I{kappa}B{alpha} binds to 14-3-3 proteins independently of TNF{alpha}. (a) Pull-down experiment using GST-14-3-3{eta} and lysates from HEK-293T cells incubated with TNF{alpha} as indicated. Upper panel shows immunoblot with {alpha}-p65 and {alpha}-I{kappa}B{alpha} antibodies. Ponceau staining of GST proteins is shown in lower panel. Inputs represent 1/10 of the lysates. (b) Immunoprecipitation with anti-HA from control HEK-293T cells and cells transfected with HA-I{kappa}B{alpha}32-36, treated for 60 minutes with TNF{alpha}. The presence of co-precipitated endogenous 14-3-3 and p65 was determined by western blot with anti-14-3-3{gamma}/{eta} or anti-p65 antibodies. (c) Pull-down assay with GST-14-3-3{eta} and cell lysates from HEK-293T cells untreated or treated with acid phosphatase for 30 minutes. The presence of I{kappa}B{alpha} proteins in the precipitates was determined by immunoblotting with anti-I{kappa}B{alpha} antibody. Ponceau staining of GST proteins is shown in the lower panel. Inputs represent 1/10 of the lysates. (d) Pull-down experiment using different GST-I{kappa}B{alpha} constructs and lysates from HEK-293T cells transfected with myc-14-3-3{eta}. Upper panel shows immunoblot with anti-myc antibody. Coomassie staining of GST proteins is shown in middle panel. (e,f) Pull down experiments using GST-14-3-3{eta} and lysates from HEK-293T cells transfected with the indicated Ha-I{kappa}B{alpha} deletion mutants. Upper panels show immunoblot with {alpha}-HA. Coomassie staining of GST proteins is shown in the lower right panels. Inputs represent 1/10 of the lysates. A schematic overview of the I{kappa}B{alpha} protein is shown in d-f, indicating the putative 14-3-3 binding domain.

 

Figure 4
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  		Fig. 4. 14-3-3 binding domains in p65 and I{kappa}B{alpha} are important to regulate their subcellular distribution. (a) GFP-p65 mutants were transfected in RPW1 cells to determine their subcellular localization. Slides from three independent experiments were counted on the BX-60 microscope and the percentage of cells displaying nuclear p65 is indicated. Representative confocal images are shown (magnification, 630x). (b) Immunolocalization of flag-p65 and the I{kappa}B{alpha} mutants in I{kappa}B{alpha}-/- MEF transfected with the indicated plasmids. Slides were counted on the BX-60 microscope and the percentage of cells displaying nuclear p65 and I{kappa}B{alpha} is indicated. Representative images are shown (magnification, 400x). (c) Cell lysates from HEK-293T cells cotransfected with HA-I{kappa}B{alpha} and the indicated GFP-p65 mutants were precipitated with anti-HA antibody. The presence of wild-type or mutant GFP-p65 in the precipitates was determined by immunoblotting with anti-p65 antibody. Input represents 1/10 of the lysates. (d) Anti-HA antibody was used to precipitate cell lysates from HEK-293T transfected with GFP-p65 and the indicated HA-I{kappa}B{alpha} mutants. Western blot {alpha}-p65 is shown in the upper panel. Input represents 1/10 of the lysates.

 

Figure 5
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  		Fig. 5. 14-3-3 activity is required to maintain p65 in the cytoplasm. (a) Immunolocalization of endogenous p65 in HEK-293T cells transfected with the indicated 14-3-3 plasmids and incubated with TNF{alpha} for 15 and 60 minutes as indicated. Representative confocal images are shown (magnification, 630x). Western blot showing the levels of transfected wild type (WT) and DN-14-3-3 (DN) with anti-myc antibody. (b) Pull-down experiment using GST-14-3-3{eta} and lysates from HEK-293T cells untransfected or transfected with DN-14-3-3 treated for 30 minutes with TNF{alpha} as indicated. Upper panels show immunoblot with anti-p65 antibody. Inputs represent 1/10 of the lysates. Ponceau staining of GST proteins is shown in lower panel. (c) Nuclear extracts from HEK-293T cells, untransfected or transfected with DN-14-3-3 and treated with TNF{alpha} at different time points, were precipitated with the anti-p65 antibody. Co-precipitated I{kappa}B{alpha} was detected by western blot with {alpha}-I{kappa}B{alpha} antibody. Tubulin was detected as a fractionation control and HDAC1 as a loading control for nuclear extracts in the left panel. Inputs represent 1/10 of the lysates. (d) Pull-down experiment using GST-14-3-3{eta} and lysates from wild-type or I{kappa}B{alpha}-knockout MEF cells untreated or treated for 30 minutes with TNF{alpha} as indicated. Upper panel show immunoblot with anti-p65 antibody. Ponceau staining of GST proteins is shown in middle panel. Inputs represent 1/10 of the lysates (detected with anti-p65). (e) Subcellular localization of endogenous p65 in HEK-293T cells treated with siRNA against different 14-3-3 isoforms. Right panels show the nuclear entry of HDAC4 in the 14-3-3-{epsilon} siRNA-treated cells as a control. Representative confocal images are shown (magnification, 630x). PI was used for nuclear staining. Western blot assayed with anti-pan-14-3-3 antibody recognizing 14-3-3 family members shows the levels of 14-3-3 in cell lysates from HEK-293T cells treated with the isoform-specific siRNA. HDAC1 was detected as a loading control.

 

Figure 6
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  		Fig. 6. 14-3-3 proteins associate with the chromatin in response to TNF{alpha} to modulate NF{kappa}B-dependent transcription. (a) Subcellular localization of endogenous 14-3-3 (left panels), p65 (middle panels) and I{kappa}B{alpha} (right panels) in control or HEK-293T cells treated for 30 minutes with TNF{alpha}. PI was used for nuclear staining. Slides from three independent experiments were counted on the BX-60 microscope and the percentage of cells displaying nuclear 14-3-3 is indicated. Representative confocal images are shown (magnification, 630x). (b) Western blot with the indicated antibodies of total cell lysates (left) and nuclei (right) from HEK-293T cells incubated with TNF{alpha} at different time points. Absence of ß-actin in the nuclear extracts is shown as a fractionation control whereas HDAC1 was detected as a nuclear loading control. (c) Chromatin from TNF{alpha}-treated NIH-3T3 cells was immunoprecipitated with anti-14-3-3ß and -{gamma}, anti-p65 and anti-I{kappa}B{alpha} antibodies. The presence of the indicated NF{kappa}B target promoters in the precipitates was determined by PCR. (d) Chromatin from HEK-293T cells untransfected or transfected with DN14-3-3 and treated with TNF{alpha} at different time points, was immunoprecipitated with the {alpha}-p65 antibody. The presence of the indicated NF{kappa}B target promoters in the precipitates was determined by PCR. (e) Semiquantitative RT-PCR to determine the transcriptional activity of the indicated NF{kappa}B target genes in HEK-293T cells untransfected ({blacktriangleup}) or transfected with DN-14-3-3 ({blacksquare}) treated with TNF{alpha} at the indicated time points. Graphs represent the relative amounts of mRNA, as measured by densitometric analysis, from one of two equivalent experiments.

 




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