STATc is a key regulator of the transcriptional response to hyperosmotic shock-2

<p><b>Copyright information:</b></p><p>Taken from "STATc is a key regulator of the transcriptional response to hyperosmotic shock"</p><p>http://www.biomedcentral.com/1471-2164/8/123</p><p>BMC Genomics 2007;8():123-123.</p><p>Published online 21 May 2007</p><p>PMCID:PMC1888708.</p><p></p>1.5 fold in the time course of sorbitol treatment was clustered with GeneSpring 7.2. Four major clusters (1–4) can be distinguished. The dendrogram is displayed on the left. The differentially regulated genes are depicted as coloured lines and the time of treatment in minutes is shown at the bottom. The colour represents the fold induction (red) or repression (blue) as shown in the colour scale below the figure. Non-regulated genes are displayed in yellow. (B) A selection of the GO biological process terms that were enriched in each of the clusters is presented. GO tree levels are shown on the left. Bar lengths represent the fold enrichment (scale x-axis). The table indicates the number of genes with a particular annotation in the cluster (List), on the entire array (Total), the significance for enrichment (P-value) and the annotation. (C) Expression profiles of selected genes from each cluster. The following abbreviations for differentially regulated genes are used. Cluster 1: A, A; B6, 20S proteasome subunit beta-6; C4, 26S proteasome subunit ATPase 4; 11, 26S proteasome non-ATPase regulatory subunit 11; 2, 26S proteasome regulatory subunit 2; C, Cysteine Protease Inhibitor; A, cysteine proteinase 1. Cluster 2: A, coronin; B, actin related protein 2; B, profilin II; A, NCK-Associated Protein; C, actin binding protein; act8, actin8. Cluster 3: C, STATc; A, RasGTPase-activating protein; A, Severin kinase; G21, ABC transporter G family protein. Cluster 4: , vacuolar H+-ATPase subunit

STATc is a key regulator of the transcriptional response to hyperosmotic shock-3

<p><b>Copyright information:</b></p><p>Taken from "STATc is a key regulator of the transcriptional response to hyperosmotic shock"</p><p>http://www.biomedcentral.com/1471-2164/8/123</p><p>BMC Genomics 2007;8():123-123.</p><p>Published online 21 May 2007</p><p>PMCID:PMC1888708.</p><p></p> if STATc is not involved in the transcriptional regulation. (B) Expected overlap in experiments I and II if STATc is the only transcriptional regulator in response to hypertonicity. (C) Venn diagram of the observed differentially regulated genes from the three comparisons: wt cells treated versus untreated (I), RIC cells treated versus STATctreated (II) and STATctreated versus untreated (III). The numbers of up- and down-regulated genes of the single experiments are printed in red and green, respectively. Genes shared between 2 or 3 comparisons (shaded region) were applied to further analysis

STATc is a key regulator of the transcriptional response to hyperosmotic shock-1

<p><b>Copyright information:</b></p><p>Taken from "STATc is a key regulator of the transcriptional response to hyperosmotic shock"</p><p>http://www.biomedcentral.com/1471-2164/8/123</p><p>BMC Genomics 2007;8():123-123.</p><p>Published online 21 May 2007</p><p>PMCID:PMC1888708.</p><p></p>ts. The data are expressed as means of fold change ± SD of three independent experiments. The corresponding DDB IDs from left to right as follows: DDB0188166, DDB0235172, DDB0188166, DDB0190245, DDB0185120

STATc is a key regulator of the transcriptional response to hyperosmotic shock-4

<p><b>Copyright information:</b></p><p>Taken from "STATc is a key regulator of the transcriptional response to hyperosmotic shock"</p><p>http://www.biomedcentral.com/1471-2164/8/123</p><p>BMC Genomics 2007;8():123-123.</p><p>Published online 21 May 2007</p><p>PMCID:PMC1888708.</p><p></p>ters (1–8) can be distinguished of which clusters 4 and 7 contain those genes that are solely regulated by STATc. The dendrogram is displayed on the left. The differentially regulated genes are depicted as coloured lines. The colour represents the fold of induction (red) or repression (blue) (colour scale see Fig. 3). Non-regulated genes are displayed in yellow. OP1: Osmostress induced pathway 1; OSP: Osmostress induced STATc pathway; SP: STATc pathway irrespective of osmostress. (B) GO biological process terms enriched in cluster 4 and 7. GO tree levels are shown on the left. Bar lengths represent the fold enrichment (scale x-axis). The table indicates the number of genes with a particular annotation in the cluster (List), on the entire array (Total), the significance for enrichment (P-value) and the annotation

Nuclear translocation of STATc is delayed in pyk3⁻, phg2⁻, and pyk3⁻/phg2⁻ cells.

<p>(A) Ax2 wild-type, pyk3⁻, phg2⁻, and pyk3⁻/phg2⁻ cells expressing GFP-STATc were treated with 100 mM sorbitol for the indicated times to induce GFP-STATc nuclear translocation, fixed with ice-cold methanol, and observed under the fluorescence microscope. Exemplary images of GFP-STATc expressing cells after 0, 3, and 8 min of treatment are shown and the insets at 3 min show a single exemplary cell for each strain to show prominent nuclear (Ax2), transition i.e. beginning nuclear (pyk3⁻ and phg2⁻) and clear cytosolic (pyk3⁻/phg2⁻) localisation of GFP-STATc. (B) Quantification of GFP-STATc nuclear translocation in Ax2 wild-type and mutant cells. For each time point, we analysed 150 cells per experiment and determined the number of cells showing either clear cytosolic (black bar), transition i.e. beginning nuclear (grey bar) or prominent nuclear (white bar) localisation of GFP-STATc. The percentage of cells in each of these three categories was calculated. Error bars depict standard deviations of two independent experiments.</p

Phg2 but not Pyk3 acts upstream of PTP3.

<p>(A) Upon hyperosmotic conditions, PTP3 becomes phosphorylated on two serine residues, S448 and S747, resulting in an inhibition of its phosphatase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090025#pone.0090025-Araki2" target="_blank">[7]</a>. (B–E) Ax2/Myc-PTP3, pyk3⁻/Myc-PTP3 and phg2⁻/Myc-PTP3 cells were left untreated (−) or treated (+) with 200 mM sorbitol for 5 min (B, C) or 10 min (D, E). Myc-PTP3 was immunoprecipitated, separated by SDS-PAGE, transferred to nitrocellulose, and phosphorylated Myc-PTP3 was detected with PTP3 antibodies specific for phospho-serine 448 (B, D) and phospho-serine 747 (C, E). Total Myc-PTP3 was used as loading control and detected with an anti-Myc antibody (mAb 9E10). (F, G): Quantification of PTP3 phospho-serine 448 (F) and 747 (G) in either treated or untreated cells. Band intensities were determined densitometrically and normalised using the values for total Myc-PTP3. The amount of serine phosphorylated PTP3 of treated Ax2/Myc-PTP3 cells was set to 1 and relative values were calculated for untreated Ax2/Myc-PTP3 cells as well as for treated and untreated phg2^−/Myc-PTP3 cells. The error bars depict standard deviations of three independent experiments. (H, I): Pull-down assay to investigate the binding of Myc-PTP3 to bacterially expressed GST-Phg2. In the first approach (H) Myc-PTP3 was expressed in phg2⁻ cells, immunoprecipitated with anti-Myc Dynabeads, and eluted from the beads via a pH shift for binding to GST-Phg2 bound to glutathione beads. Lane 1: Total cell lysate of Myc-PTP3 expressing phg2⁻ cells; lane 2: Anti-Myc Dynabeads after immunoprecipitation of Myc-PTP3; lane 3: Myc-PTP3 after elution from anti-Myc Dynabeads; lane 4: GST, purified from bacteria and bound to glutathione-beads (GST-beads); lane 5: GST-Phg2, purified from bacteria and bound to glutathione-beads (GST-Phg2-beads); lane 6: supernatant after incubation of Myc-PTP3 with GST-beads; lane 7: pellet after incubation of Myc-PTP3 with GST-beads; lane 8: supernatant after incubation of Myc-PTP3 with GST-Phg2-beads; lane 9: pellet after incubation of Myc-PTP3 with GST-Phg2-beads. In the reverse approach (I) the binding of bacterially expressed GST-Phg2, which was eluted from glutathione-beads, to Myc-PTP3 bound to anti-Myc Dynabeads (Myc-PTP3-beads) was investigated. Lane 1: Total cell lysate of Myc-PTP3 expressing phg2⁻ cells; lane 2: lysate after immunoprecipitation of Myc-PTP3 with anti-Myc Dynabeads; lane 3: Anti-Myc Dynabeads with bound Myc-PTP3; lane 4: GST, purified from bacteria and eluted from glutathione-beads; lane 5: GST-Phg2, purified from bacteria and eluted from glutathione-beads; lane 6: supernatant after incubation of GST with Myc-PTP3-beads; lane 7: pellet after incubation of GST with Myc-PTP3-beads; lane 8: supernatant after incubation of GST-Phg2 with Myc-PTP3-beads; lane 9: pellet after incubation of GST-Phg2 with Myc-PTP3-beads. The order of lines from the same immunoblot were digitally re-arranged for illustration purposes to omit dispensable lines.</p

Primer pairs used for quantitative Real-Time PCR analysis.

<p>Oligonucleotide primers were designed on the basis of sequence information and purchased from Metabion Corp. (Munich, Germany).</p

Real time PCR confirms STATc-dependent transcriptional activation of selected genes.

<p>The differential expression of seven selected genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090025#pone-0090025-t003" target="_blank">Table 3</a>) in Ax2 wild-type (black bars) and STATc⁻ (white bars) cells in response to treatment with 200 mM sorbitol for 15 minutes was analysed by quantitative real time PCR. The data are expressed as means of fold change in comparison to untreated cells. Fold changes and standard deviations of six measurements from three independent experiments are shown. N/A: <i>not applicable.</i></p

Model of STATc activation in response to hyperosmotic conditions.

<p>The model is based on results from previously published studies (green) and from results (blue) and educated assumptions (red) of the present work. In response to hyperosmolarity intracellular cGMP and Ca²⁺ act in parallel as second messengers in order to activate STATc. GbpC is downstream of cGMP and we postulate that it acts upstream of Pyk3 and an as yet unidentified STATc protein kinase (SPK). Phg2 inhibits PTP3 either directly or indirectly by phosphorylation of S747 and an unknown additional PTP3 serine/threonine protein kinase (PPK) must be responsible for phosphorylation of PTP3 at S448. These protein kinases are under control of the Ca²⁺ branch of the STATc signaling cascade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090025#pone.0090025-Araki2" target="_blank">[7]</a>. The model does not satisfactorily explain all experimental results and we additionally propose a crosstalk between the two signaling branches (not shown). See the Discussion section for further details of this model.</p

<i>D. discoideum</i> mutant strains used in this study.

<p><i>D. discoideum</i> mutant strains used in this study.</p