12 research outputs found
Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation.
Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions
Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation
4‑Methylumbelliferone-Functionalized Polyphosphazene and Its Assembly into Biocompatible Fluorinated Nanocoatings with Selective Antiproliferative Activity
Advanced multifunctional biomaterials are increasingly
relying
on clinically dictated patterns of selectivity against various biological
targets. Integration of these frequently conflicting features into
a single material surface may be best achieved by combining various
complementary methodologies. Herein, a drug with a broad spectrum
of activity, i.e., 4-methylumbelliferone (4-MU),
is synthetically multimerized into water-soluble anionic macromolecules
with the polyphosphazene backbone. The polymer structure, composition,
and solution behavior are studied by 1H and 31P NMR spectroscopy, size-exclusion chromatography, dynamic light
scattering, and UV and fluorescence spectrophotometry. To take advantage
of the clinically proven hemocompatibility of fluorophosphazene surfaces,
the drug-bearing macromolecule was then nanoassembled onto the surface
of selected substrates in an aqueous solution with fluorinated polyphosphazene
of the opposite charge using the layer-by-layer (LbL) technique. Nanostructured
4-MU-functionalized fluoro-coatings exhibited a strong antiproliferative
effect on vascular smooth muscle cells (VSMCs) and fibroblasts with
no cytotoxicity against endothelial cells. This selectivity pattern
potentially provides the opportunity for highly desirable fast tissue
healing while preventing the overgrowth of VSMCs and fibrosis. Taken
together with the established in vitro hemocompatibility and anticoagulant
activity, 4-MU-functionalized fluoro-coatings demonstrate potential
for applications as restenosis-resistant coronary stents and artificial
joints
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
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
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
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
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
Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70 T293
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