Casodex and S1 peptide inhibit EGF-stimulated BrdU incorporation and migration in HCT116 and KP-2 cells.

<div><p>Quiescent human colon cancer-derived HCT116 cells were used. In <b>A</b>, cells on coverslips were left un-stimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After <i>in </i><i>vivo</i> pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In <b>B</b>, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments. </p> <p>Quiescent human pancreatic cancer-derived KP-2 cells were used. In <b>C</b>, cells on coverslips were left unstimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After <i>in </i><i>vivo</i> pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In <b>D</b>, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments. </p> <p>In <b>A</b>, B, C and <b>D</b>, (*) <i>p</i> value < 0.001; (**) p value < 0,005. </p> <p>Panel <b>E</b> shows the Western blot of HCT116 or KP-2 cell lysates with the antibodies against the indicated proteins: tubulin, epidermal growth factor receptor (EGFR) and androgen receptor (AR).</p></div

Inhibition by the S1 peptide of AR/Src complex, Src activation and DNA synthesis triggered by EGF in HT1080 cells.

<p>Quiescent HT1080 cells were used. Cells were left un-stimulated or stimulated for 10 min with EGF (at 100 ng/ml) in the absence or presence of S1 or Ss peptide (both at 1 nM). Casodex (at 10 μM) was used for comparison with the S1 peptide. Upper section in <b>A</b>, Western blot of HT1080 cell lysates with anti-EGFR antibody. Tubulin (tubulin) was revealed by immunoblot, as a loading control. Lower section in <b>A</b>, lysates were immune-precipitated with anti-EGFR Ab and proteins in immune complexes were detected using antibodies against the indicated proteins. In <b>B</b>, lysates were immune-precipitated with the anti-Src MAb and Src activity in immune complexes was assayed using acidified enolase, as a substrate. In <b>C</b>, cells on coverslips were left untreated or treated for 18 h with the indicated compounds. After <i>in </i><i>vivo</i> pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total nuclei. Several independent experiments were performed in duplicate and the results were obtained from at least 500 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. (*) p value < 0,001; (°) p value< 0,005.</p

Characterization of the p300 Taz2–p53 TAD2 Complex and Comparison with the p300 Taz2–p53 TAD1 Complex

The p53 tumor suppressor is a critical mediator of the cellular response to stress. The N-terminal transactivation domain of p53 makes protein interactions that promote its function as a transcription factor. Among those cofactors is the histone acetyltransferase p300, which both stabilizes p53 and promotes local chromatin unwinding. Here, we report the nuclear magnetic resonance solution structure of the Taz2 domain of p300 bound to the second transactivation subdomain of p53. In the complex, p53 forms an α-helix between residues 47 and 55 that interacts with the α1−α2−α3 face of Taz2. Mutational analysis indicated several residues in both p53 and Taz2 that are critical for stabilizing the interaction. Finally, further characterization of the complex by isothermal titration calorimetry revealed that complex formation is pH-dependent and releases a bound chloride ion. This study highlights differences in the structures of complexes formed by the two transactivation subdomains of p53 that may be broadly observed and play critical roles in p53 transcriptional activity

Alternatively activated p38 uniquely phosphorylates NFAT1 on S79.

<p>(A) In vitro kinase assay in which recombinant mouse p38α was incubated with active human zeta-associated protein (ZAP-70), mitogen-activated protein kinase kinase 6 (MKK6), or buffer alone. After 1 hour, recombinant ATF2 (left panel) or tNFAT1 (right panel) and 10 μCi [³²P]ATP were added for 30 minutes before separation on SDS-PAGE and PhosphorImager analysis. The results are representative of 3 independent experiments. (B) Recombinant mouse p38α was incubated with active human ZAP-70, MKK6, or buffer alone in in vitro kinase buffer. After 1 hour, recombinant tNFAT1 was added and incubated for an additional hour before separation on SDS-PAGE and immunoblotting with antibodies specific for pNFAT1^S79. (C) Freshly purified wild-type (WT) T cells were stimulated with anti-CD3/CD28, phorbol myristate acetate (PMA)/ionomycin, or buffer alone for 15 minutes. Cells were stained for total NFAT1 (green) or pNFAT1^S79 (red) and imaged by confocal microscopy. Scale bar = 250 pixels. (D) Quantification of the percent of cells positive for NFAT1^pS79 (left panel) and percent of cells with nuclear NFAT1 (right panel) per high power field (HPF) in the experiment shown in panel C. Each dot represents an individual HPF (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s003" target="_blank">S3 Data</a>). ****<i>p</i> < 0.0001. NS, not significant.</p

Phosphorylation of NFAT1^S79A is required for nuclear migration upon T-cell antigen receptor (TCR) stimulation.

<p>(A) Purified T cells from wild-type (WT) mice were infected with retrovirus encoding HA-NFAT1 or HA-NFAT1^S79A. The cells were stimulated with anti-CD3/CD28 for 1 hour and examined for NFAT1 (red) localization by confocal microscopy. DAPI was used to stain the nucleus. Scale bar = 10 μM. (B) Purified primary T cells from WT mice were stimulated and infected as in panel A, and NFAT1 levels in the cytosolic and nuclear fractions were assessed by immunoblotting. (C) Stable Jurkat cell lines expressing HA-NFAT1 or HA-NFAT1^S79A were stimulated with anti-CD3/CD28 for 1 hour, and the lysates were immunoprecipitated (IP) with anti-HA and immunoblotted (IB) for calcineurin A and HA. (D) Confocal images of in situ proximity ligation assay (PLA) of stable Jurkat cell clones expressing HA-NFAT or HA-NFAT1^S79A that had been stimulated with anti-CD3/CD28 for 15 minutes. Alexa Fluor 488 (green)-conjugated wheat germ agglutinin (WGA) was used to stain plasma membrane. Scale bar = 100 pixels (left panel). Quantification of the average dots and intensity per cell (WT-Uns [<i>n</i> = 166], anti-CD3/CD28 [<i>n</i> = 132]; S79A-Uns [<i>n</i> = 162], and anti-CD3/CD28 [<i>n</i> = 132]) (right panel) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s005" target="_blank">S5 Data</a>). **<i>p</i> < 0.01, ****<i>p</i> < 0.0001. NS, not significant.</p

Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn²⁺ or Mg²⁺ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn²⁺ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg²⁺ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange–mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg²⁺, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30–300 s. Submillimolar Mg²⁺ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192–200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg²⁺ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg²⁺ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain

The p38 alternative pathway is required for NFAT1 nuclear migration.

<p>(A) Purified T cells from wild-type (WT) or double knock-in (DKI) mice were stimulated with anti-CD3/CD28 or phorbol myristate acetate (PMA)/ionomycin for 3 hours, and NFAT1 levels in the nuclear and cytosolic fractions were determined by immunoblotting. (B) Purified T cells from WT or DKI mice were stimulated as in panel A, and NFAT1 (red) localization was visualized by confocal microscopy. DAPI (blue) was used to stain the nucleus. Scale bar = 10 μM. (C) Quantification of the percentage of cells with nuclear NFAT1 per high power field (HPF). Error bars are the mean ± SEM (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s002" target="_blank">S2 Data</a>). *<i>p</i> < 0.05, ***<i>p</i> < 0.001. NS, not significant.</p

Role of NFAT1 and NFAT1^S79 in NFAT2 and cytokine expression.

<p>(A) Interleukin (IL)-2 and tumor necrosis factor alpha (TNF-α) production in supernatants of wild-type (WT) or N1KO Jurkat clones stimulated with anti-CD3/CD28, phorbol myristate acetate (PMA)/ionomycin, or medium alone for 20 hours. The results represent the mean of 3 independent experiments ± SEM (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s004" target="_blank">S4 Data</a>). (B) WT or N1KO Jurkat clones were stimulated with anti-CD3/CD28, PMA/ionomycin, or medium alone for 48 hours, and NFAT2 expression was determined by immunoblotting. (C) The N1KO Jurkat clone was infected with retrovirus encoding HA-NFAT or HA-NFAT1^S79A, followed by single cell sorting of green fluorescent protein-positive (GFP⁺) cells. Quantitation of transduced gene product expression in 2 independent clones from each transduction was determined by immunoblotting with anti-HA. (D) Quantitation of IL-2 in the supernatants of HA-NFAT1 or HA-NFAT1^S79A Jurkat clones stimulated with anti-CD3/CD28, PMA/ionomycin, or medium alone. The results represent the mean of 3 independent experiments ± SEM (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s004" target="_blank">S4 Data</a>).</p

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator