Fig 1.
p38 Alternative activation is required for T-cell antigen receptor (TCR)-induced expression of NFAT2 via c-Fos.
(A, left panel) Purified mouse T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and phorbol myristate acetate (PMA) plus ionomycin for 16 hours and then immunoblotted. (A, right panel) Purified mouse T cells were stimulated as in panel A, and interleukin (IL)-2 was measured in the culture supernatant (S1 Data). (B) Purified T cells from wild-type (WT) mice were stimulated with anti-CD3/CD28 or PMA/ionomycin for the indicated times, and then the lysates were immunoblotted. (C) Purified T cells from WT or double knock-in (DKI) mice were stimulated with anti-CD3/CD28 for the indicated times, and then the lysates were immunoblotted. (D) Purified WT T cells were stimulated with anti-CD3/CD28 or PMA/ionomycin for the indicated times, and binding of c-Fos to the NFAT2 promoter was analyzed by chromatin immunoprecipitation (ChiP) (S1 Data). (E) pGL3-basic luciferase reporter construct containing the nfat2 promoter consensus AP-1 binding site or its mutant (left panel) was transfected into Jurkat cells, which were stimulated overnight with anti-CD3/CD28, and then a luciferase assay was performed (right panel) (S1 Data). (F) Purified T cells from 2 WT or 2 DKI mice were infected with retrovirus carrying empty vector (EV) or c-fos. The T cells were pooled per group and stimulated with anti-CD3/CD28 for 24 hours, and the expression of nfat2 mRNA was determined by quantitative real-time PCR. nfat2 levels in WT EV-transduced and unstimulated samples were defined as 1 (S1 Data). (G) Purified murine T cells were infected and stimulated with anti-CD3/CD28 or PMA/ionomycin, and nfat2 mRNA expression was quantitated by real-time PCR as in panel F (S1 Data). The results are representative of 3 independent experiments, and the bar graphs in the right panel show the mean ± SEM of all 3. *p < 0.05, **p <0.01, ***p < 0.001. NS, not significant.
Fig 2.
The p38 alternative pathway is required for NFAT1 nuclear migration.
(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 (S2 Data). *p < 0.05, ***p < 0.001. NS, not significant.
Fig 3.
Alternatively activated p38 uniquely phosphorylates NFAT1 on S79.
(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 pNFAT1S79. (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 pNFAT1S79 (red) and imaged by confocal microscopy. Scale bar = 250 pixels. (D) Quantification of the percent of cells positive for NFAT1pS79 (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 (S3 Data). ****p < 0.0001. NS, not significant.
Fig 4.
Role of NFAT1 and NFAT1S79 in NFAT2 and cytokine expression.
(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 (S4 Data). (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-NFAT1S79A, 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-NFAT1S79A Jurkat clones stimulated with anti-CD3/CD28, PMA/ionomycin, or medium alone. The results represent the mean of 3 independent experiments ± SEM (S4 Data).
Fig 5.
Phosphorylation of NFAT1S79A is required for nuclear migration upon T-cell antigen receptor (TCR) stimulation.
(A) Purified T cells from wild-type (WT) mice were infected with retrovirus encoding HA-NFAT1 or HA-NFAT1S79A. 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-NFAT1S79A 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-NFAT1S79A 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 [n = 166], anti-CD3/CD28 [n = 132]; S79A-Uns [n = 162], and anti-CD3/CD28 [n = 132]) (right panel) (S5 Data). **p < 0.01, ****p < 0.0001. NS, not significant.