6 research outputs found
Phosphorylation of NFAT1<sup>S79A</sup> 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<sup>S79A</sup>. 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<sup>S79A</sup> 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<sup>S79A</sup> 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
Role of NFAT1 and NFAT1<sup>S79</sup> 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<sup>S79A</sup>, followed by single cell sorting of green fluorescent protein-positive (GFP<sup>+</sup>) 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<sup>S79A</sup> 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
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 [<sup>32</sup>P]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<sup>S79</sup>. (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<sup>S79</sup> (red) and imaged by confocal microscopy. Scale bar = 250 pixels. (D) Quantification of the percent of cells positive for NFAT1<sup>pS79</sup> (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
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
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
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