18 research outputs found

    NKX3.1 expression and interactions Dataset

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    <p>Data set 1: Summary of NKX3.1 interacting proteins</p> <p>Data set 1A: All proteins identified in four independent mock and FLAG-NKX3.1 affinity purifications<br>Data set 1B: List of 58 high confidence NKX3.1 interacting proteins<br>Data set 1C: Lists of proteins shown in the Venn diagram in Fig. 1B</p> <p>Data set 2: Summary of NKX3.1-regulated gene expression</p> <p>Data set 2A: All raw mRNA expression data<br>Data set 2B: Averaged mRNA expression data<br>Data set 2C: List of mRNAs that show a greater than 5-fold change (p greater or even to 0.05) in cells expressing NKX3.1 for 7 hours (= "5x data set")<br>Data set 2D: list of NKX3.1 regulated genes containing conserved AP1 binding sites<br>Data set 2E: List of NKX3.1 regulated genes containing conserved SRF binding sites<br>Data set 2F: List of NKX3.1 regulated mRNAs that are inversely regulated in human prostate cancer derived cell lines</p> <p> </p

    FGFR2 interacts with IKKβ and stimulates tyrosine phosphorylation of IKKβ.

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    <p>FGFR2 wildtype (WT) or kinase dead (KD) and IKKβ were expressed in HEK293 cells. (<b>A</b>) <b>FGFR2 associates with IKKβ</b>. IKKβ was immunoprecipitated from lysates and analysed for FGFR2 by immunoblot (top panel). The membrane was stripped and reprobed for IKKβ (second panel). Expression of FGFR2 and IKKβ is shown in cell lysates (lower panels). (<b>B</b>) <b>IKKβ associates with FGFR2</b>. FGFR2 was immunoprecipitated from lysates and analysed for IKKβ by immunoblot (top panel). The membrane was stripped and reprobed for FGFR2 (second panel). Lysate blots are as in (A). (<b>C</b>) <b>FGFR2 and IKKβ are present in complexes with IKKγ/NEMO</b>. Endogenous IKKγ/NEMO was immunoprecipitated from cell lysates expressing FGFR2 and IKKβ using IKKγ/NEMO antisera. The interaction with FGFR2 (top panel) and IKKβ (second panel) was detected by immunoblot. Negative IgG control shown in lane 4B. Note that the 1<sup>st</sup> and 2<sup>nd</sup> panels represent duplicate gels of the same samples. The thin black lines on the 2<sup>nd</sup> panel indicate where additional IgG controls were run but removed from the final figure except for Lane 4B. All samples on this panel are from the same exposure of the same immunoblot. Expression of FGFR2 and IKKβ shown in total lysate (lower panels). (<b>D</b>) <b>FGFR2 stimulates tyrosine phosphorylation of IKKβ</b>. IKKβ was immunoprecipitated from lysates and analysed by phophotyrosine immunoblot (top panel). The membrane was stripped and reprobed for IKKβ (second panel). Expression of FGFR2 derivatives and IKKβ is shown in total lysates (lower panels). The arrow in Lane 5 of the upper panel indicates IKKβ.</p

    Tyrosine Phosphorylation Allows Integration of Multiple Signaling Inputs by IKKβ

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    <div><p>Signaling regulated by NFκB and related transcription factors is centrally important to many inflammatory and autoimmune diseases, cancer, and stress responses. The kinase that directly regulates the canonical NFκB transcriptional pathway, Inhibitor of κB kinase β (IKKβ), undergoes activation by Ser phosphorylation mediated by NIK or TAK1 in response to inflammatory signals. Using titanium dioxide-based phosphopeptide enrichment (TiO<sub>2</sub>)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), we analyzed IKKβ phosphorylation in human HEK293 cells expressing IKKβ and FGFR2, a Receptor tyrosine kinase (RTK) essential for embryonic differentiation and dysregulated in several cancers. We attained unusually high coverage of IKKβ, identifying an abundant site of Tyr phosphorylation at Tyr169 within the Activation Loop. The phosphomimic at this site confers a level of kinase activation and NFκB nuclear localization exceeding the iconic mutant S177E/S181E, demonstrating that RTK-mediated Tyr phosphorylation of IKKβ has the potential to directly regulate NFκB transcriptional activation. </p> </div

    Composite analysis of mutations of phospho-acceptor sites within the

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    <p>IKKβ <b><i>Acitvation </i></b><b><i>Loop</i></b>. (<b>A</b>) <b>Contribution of Tyr169, Ser177, Thr180, and Ser181 to IKKβ kinase activation</b>. All possible combinations of single and double mutations were constructed in the IKKβ Activation Loop phospho-acceptor sites, Ser177 and Ser 181, identified previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084497#B24" target="_blank">24</a>], and Tyr169 and Thr180 identified in this work. Immunoprecipitated IKKγ/NEMO complexes from HEK293 cells were assayed for <i>in </i><i>vitro</i> kinase activity against the substrate GST-IκBα<sup>(1-54)</sup> (top panel), and IKKβ expression is shown (lower panel). (<b>B</b>) <b>Requirement for multiple hydroxyl amino acids within Activation Loop</b>. Multiple mutations within the Activation Loop probe minimal requirements for activation. Mutations were constructed within the Activation Loop phospho-acceptor sites to examine whether Y169E could provide activation when combined with the mutations S177A, T180A, and S181A (compare Lanes 3 and 4). Similarly, Lanes 5 and 6 examine the ability of the “EE” mutations S177E/S181E to provide activation when combined with Y169F and T180A. IKKγ/NEMO immunoprecipiates were examined for <i>in </i><i>vitro</i> kinase activity against the substrate GST-IκBα<sup>(1-54)</sup> (top panel). IKKβ expression is shown (lower panel). (<b>C</b>) <b>Y169E stimulates S177/S181 phosphorylation</b>. Activation Loop phosphorylation detected using phospho-S177/S181 antiserum. The ability of IKKβ WT and Y169F to stimulate phosphorylation of S177/S181 as detected by phospho-specific immunoblotting is presented, in comparison with the lack of activity shown by the S177E/S181E “EE” and Y169F mutants. IKKβ expression is shown (lower panel). </p

    Effect of tyrosine mutations on IKKβ activation and NFκB nuclear localization.

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    <p>(<b>A</b>) <b>Effects on kinase activity of mutations at sites of IKKβ tyrosine phosphorylation</b>. Each tyrosine residue detected as phosphorylated by MS/MS analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084497#pone-0084497-t001" target="_blank">Table 1</a>) was mutated either to phenylalanine (F) to prevent phosphorylation, or to glutamic acid (E) as the phosphomimic. Indicated mutants were expressed in HEK293 cells, and resulting IKKγ/NEMO immunoprecipitates were subjected to <i>in </i><i>vitro</i> kinase assays utilizing the substrate GST-IκBα<sup>(1-54)</sup> as described in Materials and Methods. <sup>32</sup>P incorporation on GST-IκBα<sup>(1-54)</sup> (top panel) and expression of IKKβ mutant proteins in cell lysates is shown (lower panel). Quantification of the relative <sup>32</sup>P incorporation on GST-IκBα<sup>(1-54)</sup> is presented in the bar graph below, showing the standard error of the mean for a minimum of 3 independent repeats, and normalized to the activity of the S177E/S181E “EE” mutant as 100%. All IKKβ mutants presented here were untagged. (<b>B</b>) <b>Y169E promotes NFκB nuclear localization</b>. NFκB nuclear localization in response to IKKβ mutants. Indicated IKKβ proteins were expressed in MCF7 cells. Cells were fractionated as described in Materials and Methods. Cytoplasmic and Nuclear lysate fractions were immunoblotted for NFκB (p65) (top panels) and β-Tubulin (bottom panels). The top membrane was stripped and reprobed for expression of IKKβ (middle panels). Quantification of NFκB nuclear localization is presented in the bar graph below, showing the standard error of the mean for a minimum of 3 independent repeats, normalized to the activity of the S177E/S181E “EE” mutant as 100%. </p

    Mass Spectrometry analysis identifies novel phospho-acceptor sites on IKKβ.

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    <p>(<b>A</b>) <b>IKKβ schematic showing tyrosine residues</b>. A schematic of IKKβ is shown with the N-terminal kinase domain, the ubiquitin-like domain (ULD), the scaffold/dimerization domain (SDD) which also contains the leucine zipper (LZ) and helix-loop-helix (HLH) regions, and the NEMO binding domain (NBD) at the C-terminus of IKKβ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084497#B35" target="_blank">35</a>]. The location of all IKKβ tyrosine residues is shown. Tyrosine residues identified as phosphorylated by TiO<sub>2</sub>-LC-MS/MS analysis are indicated in Red (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084497#pone-0084497-t001" target="_blank">Table 1</a>). (<b>B</b>) <b>Major P-Tyr sites identified by MS/MS</b>. HEK293 cells expressing FGFR2 and IKKβ were collected in RIPA Lysis Buffer, and IKKβ immunoprecipitates were collected, treated with trypsin or pepsin, and resulting peptides prepared for MS/MS analysis as described in Materials and Methods. Spectral counts of major phospho-Tyr residues detected are presented in this graph as a percentage of the total number of phospho-Tyr peptide spectra detected for IKKβ in this study (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084497#pone-0084497-t001" target="_blank">Table 1</a>). (<b>C</b>) <b>Major P-Thr sites identified by MS/MS</b>. Spectral counts of major phospho-Thr residues detected are presented in this graph as a percentage of the total number of phospho-Thr peptide spectra detected for IKKβ in this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084497#pone.0084497.s004" target="_blank">Table S2</a>). (<b>D</b>) <b>Analysis of pY169</b>. A definitive MS/MS spectrum of the peptide containing phospho-Tyr169 is presented. (<b>E</b>) <b>Analysis of pT180</b>. A definitive MS/MS spectrum of the peptide containing phospho-Thr180 is presented. </p

    Prolyl hydroxylation of ATF4 on aa 60 and 235 by PHD1/3 limits ATF4 availability.

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    <p>(A–B) Specificity of shRNA used to KD PHD1/3. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with scrambled control or PHD1 shRNA and PHD3 shRNA alone or in combination, and treated with TM (1 µg/ml) for 6 h. The relative mRNA levels of PHD1 (A) and PHD3 (B) were determined by qPCR. The results are shown as the mean values ± S.E. of three independent experiments. Three independent shRNA were used to confirm the changes shown. (C) PHD1 and PHD3 cooperation is required and mediate the effect of Siah1a/2 on TM-induced CHOP transcription. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with different PHD1 shRNA or PHD3 shRNA, or their combination, or scrambled shRNA. Cells were treated with TM (1 µg/ml) and collected 6 h later. The relative transcription levels of CHOP were determined by qPCR. (D) PHD1 and PHD3 mediate the effect of Siah1a/2 on hypoxia-induced CHOP transcription. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with PHD1 shRNA and PHD3 shRNA alone or in combination, or control vector and exposed to 1% O<sub>2</sub> for 6 h. The relative mRNA level of CHOP was determined by qPCR. (E) PHD3 protein is induced in <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells. WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were exposed to 1% O<sub>2</sub> for 24 h prior to the analysis for the expression of HIF-1α, PHD3 and β-actin by Western blotting. The arrow points to the position of the endogenous PHD3 protein. (F) Annotated MS/MS spectra resulting in the identification of proline hydroxylation sites at P60 and P235. Identified fragment ions are shown, as are the detected sites of peptide backbone cleavage; <i>m</i>/<i>z</i>, mass to charge ratio. Note that site determining fragment ions resulted in localization of both sites of proline hydroxylation. (G) Mutations of the two identified proline hydroxylation sites at P235 and P60 to alanine stabilize ATF4 protein. 293T cells were transfected either with Flag-ATF4 or Flag-ATF4 presenting either a mutation at P60, P235, or both. After 24 h from transfection cells were treated overnight with vehicle, DMOG (0.5 mM; upper panel), or MG132 (5 µM; lower panel) followed by cell harvest and immunoblot analysis of ATF4 and β-actin. *** p<0.0005, ** p<0.005, * p<0.05 compared to ad shRNA scr. (A–D) in the same condition (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p

    Siah1/2-dependent gene expression analysis confirms an ER stress signature.

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    <p>(A) Heat map showing genes involved in diabetes, metabolism and ROS functions (light blue bar), hypoxia and related signaling (brown bar), and pathogen infection and related signaling (blue bar). Each bar represents a block of significant genes, which is involved in the same functional group. The selected genes have p-values less than or equal to 0.05 and fold-changes are greater than or equal to 2 (both directions). In the heatmap, the normalized expression signals are shown from green to red (lower signal to higher signal). The bars on the left of the heatmap indicate the functional groups. Each bar represents a block of significant genes, which involved in the same functional group. (B–C) Venn diagram representing the overlaps of significantly down- (green), and up- (red) expressed genes involved in the functional groups as indicated in the heatmap. Panel B shows genes from pairwise comparisons: knock out glucose deprivation (koGD) versus wtGD, ko oxygen deprivation (koOD) versus wtOD and ko oxygen and glucose deprivation (koOGD) versus wtOGD. Panel c shows genes from pairwise comparisons: ko versus wt, koTG versus wtTG and koTM versus wtTM. (D) Principal component analysis (PCA) 3D plot for the microarray data set. Each spot represents an individual array, and is colored based on treatment group. (E–H) Validation of representative genes from each of the main pathways identified to be Siah1/2-dependent. Siah WT and <i>Siah1a<sup>+/−</sup>::Siah2<sup>−/−</sup></i> (double knock out; DKO) MEF cells were subjected to OGD for 12 h. The relative transcription level was determined by qPCR. Shown are representative genes of the hypoxia (panel E and F), and diabetes/metabolism (panel G and H) pathways found to exhibit increase or decrease expression upon KO of Siah1a/2. The results are shown as the mean values ± S.E. of three independent experiments. (I–K) RNA prepared from Siah1a/2 WT and Siah DKO MEF cells was used for qPCR analysis. The expression of the transcripts from HSPA5 (I), Rab6 (J), GOLT1b (K) were validated by real time qPCR analysis. The results are shown as the mean values ± S.E. of three independent experiments.</p

    Siah1/2 transcription is induced by ATF4 and sXBP1 upon UPR.

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    <p>(A,B) ER-stress induction of both Siah1 and Siah2 mRNA is attenuated in <i>Atf4<sup>−/−</sup></i> MEFs (A) and <i>Ire1α<sup>−/−</sup></i> MEFs (B). Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml) or TG (1 µM) for 6 h and the relative expression of Siah1 and Siah2 mRNA was measured by qPCR. (C) IRE1α is required for ER-induced Siah2 mRNA levels. Ectopic expression of sXBP1 restores TM-induction of Siah2 mRNA in <i>Ire1α<sup>−/−</sup></i> MEFs. <i>Ire1α<sup>−/−</sup></i> MEFs were infected either with adenovirus encoding either β-gal or sXBP1. After 24 h, RNA was prepared and quantified using qPCR for the relative levels of Siah2 mRNA. (D) ER-stress induction of Siah2 mRNA levels but not of Siah1 mRNA are attenuated in <i>Atf6α<sup>−/−</sup></i> MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml) TG (1 µM) for 6 h and the relative expression of Siah1 and Siah2 mRNAs were measured by qPCR. (E) ER Stress induction of Siah1/2 transcripts is not HIF1-dependent. WT and HIF1α KO MEFs were subjected to TM (2 µg/ml) or TG (1 µM) treatment and RNA prepared 6 h later was subjected to analysis of Siah1 or Siah2 transcripts. *** p<0.0005, ** p<0.005, * p<0.07 compared to Ire1 KO (C) or to WT under the same condition (student's t test). The results are shown as the mean values ± S.E. of three independent experiments.</p
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