PMC

A Valine to Serine Point Mutation in the SP-3 Motif of NFAT4 Decreases Nuclear Export Rate
(A) The upper panel compares the sequence of the SP-3 region between NFAT1 and NFAT4. Asterisks denote phosphorylated serine residues in NFAT1 (S270, 274, 278, 282). Conserved serines are underlined. Residues in bold were mutated.
(B) Images compare nuclear dynamics of NFAT4-GFP and V-S-NFAT4-GFP, following CRAC channel activation.
(C) The graph compares nuclear import and export between V-S-NFAT4-GFP and wild-type NFAT4-GFP. Each point is the average (mean ± SEM) of six cells.
(D) Images compare nuclear efflux of wild-type NFAT4-GFP with the double mutant (V-S, S-A-NFAT4-GFP).
(E) Mean data ± SEM from eight cells for each condition are compared.
(F) Images compare nuclear dynamics of wild-type NFAT1-GFP with the S276V-NFAT1-GFP mutant.
(G) Aggregate data (mean ± SEM) from 11 NFAT1-GFP and 14 S276V-NFAT1-GFP cells are compared.
In panels (C), (E), and (G), thapsigargin was applied immediately after obtaining resting images.

NFAT Isoforms Exhibit Different Activation Thresholds
(A) Numerous Ca²⁺ oscillations are evoked by 50 nM LTC₄ in cells expressing NFAT1-GFP.
(B) A similar pattern of Ca²⁺ oscillations is induced by LTC₄ in cells expressing NFAT4-GFP.
(C) Isoform accumulation within the nucleus is compared following stimulation with LTC₄.
(D) Data (mean ± SEM) are compared. Each point represents between 6 and 14 cells.
(E–H) Identical profile to (A)–(D), but now in the presence of 20 nM LTC₄.
(I–L) The effects of 5 nM LTC₄ on NFAT1 and NFAT4 are compared, as in (A)–(D).
(M) Cytoplasmic Ca²⁺ oscillations to 2 nM LTC₄ are compared between cells expressing NFAT1-GFP (black trace) and NFAT4-GFP (red trace).
(N) Images compare NFAT1-GFP and NFAT4-GFP nuclear accumulation following stimulation with 2 nM LTC₄.
(O) Aggregate data are compared. Each point is the average of between 14 and 21 cells.
(P) The dependence of NFAT1 and NFAT4 nuclear accumulation on agonist concentration is compared. Aggregate data are mean ± SEM.

Different NFAT Isoforms Exhibit Distinct Nuclear Export Kinetics
(A) Images show the time course of NFAT1-GFP migration into the nucleus following stimulation with 2 μM thapsigargin in external solution containing 2 mM Ca²⁺. After 30 min stimulation, cells were exposed to Ca²⁺-free solution containing 0.1 mM EGTA and 1 μM cyclosporine A (depicted as EGTA+CsA), conditions that result in unidirectional NFAT export from the nucleus ().
(B) Nuclear dynamics of NFAT4-GFP are shown, under identical conditions to those described in (A).
(C) Aggregate data are compared. Each point is the average of between 17 and 22 individual cells and is represented as mean ± SEM.
(D) Gel shifts compare the extent of NFAT1, NFAT4, and (SP-3NFAT1)NFAT4-GFP rephosphorylation following stimulation with thapsigargin for 30 min to induce dephosphorylation. Rephosphorylation was then promoted by exposing the cells to Ca²⁺-free solution and cyclosporine A for 20 min, after which cell lysates were obtained. Similar results were obtained in two further independent experiments.

The SP-3 Motif of NFAT1 Slows Nuclear Export
(A) Images compare NFAT4-GFP nuclear export kinetics between a control cell and one pre-exposed to harmine (5 μM for 10 min). Aggregate data are summarized on the right. Each point shows the mean ± SEM of between 14 and 21 cells.
(B) NFAT4-GFP nuclear accumulation is compared between a control cell and one pre-exposed to harmine. Both cells were loaded with EGTA-AM prior to stimulation. The graph depicts data (mean ± SEM) from between 11 and 19 cells per point.
(C) Images compare nuclear dynamics of NFAT4-GFP with (SP-3N1)N4, which denotes NFAT4-GFP but now contains the SP-3 domain of NFAT1 instead ((SP-3N1)NFAT4-GFP). Each point in the graph is the mean ± SEM of between 14 and 24 cells.
(D) Nuclear dynamics of NFAT4-GFP and (SP-3N1)NFAT4-GFP are compared in cells loaded with EGTA. Each point on the graph shows mean data (±SEM) from between 7 and 11 cells.
(E) Nuclear movement of NFAT4-GFP is compared with (SP-3N1)N4-GFP in cells expressing PV-NLS. Each data point in the graph depicts mean ± SEM from between 14 and 23 cells.
(F) Nuclear dynamics of NFAT4-GFP and (SP-3N1)N4-GFP are compared in cells expressing PV-NES. Each point reflects mean data ± SEM from between 16 and 25 cells. In (A) and (C), NFAT movement into the nucleus was triggered by stimulating cells with thapsigargin in 2 mM external Ca²⁺ for 30 min before nuclear export was induced by perfusing cells with Ca²⁺-free external solution containing cyclosporine A, for the times shown.
In (B), (D), (E), and (F), cells were stimulated with thapsigargin in the continuous presence of external Ca²⁺.

NFAT Nuclear Dynamics in Cells Co-expressing NFAT1 and NFAT4
(A) Confocal images compare NFAT1-GFP and NFAT4-cherry distribution in the same cells. Rest denotes the non-stimulated condition, Thap. represents stimulation with thapsigargin for 30 min, and Thap. then EGTA+CsA denotes stimulation with thapsigargin for 30 min followed by exposure to thapsigargin- and Ca²⁺-free solution supplemented with cyclosporine A for 20 min prior to fixing. Nuclei were stained with DAPI.
(B) Aggregate data from experiments as in (A) are described. Each bar represents data (mean ± SEM) from at least 28 cells from 3 coverslips. N1 and N4 denote NFAT1 and NFAT4.
(C) Live cell imaging of NFAT nuclear accumulation is compared in a single cell co-expressing NFAT1-GFP and NFAT4-cherry. Thapsigargin was applied for the times indicated.
(D) NFAT nuclear export is compared in a cell co-expressing NFAT1-GFP (top) and NFAT4-cherry. After 30 min stimulation with thapsigargin, cells were perfused with Ca²⁺-free solution containing 0.1 mM EGTA and cyclosporine A.
(E) A similar experiment to that in (D) is shown, but now the stimulus was 120 nM LTC₄.
(F) Aggregate data for cells co-expressing NFAT1-GFP and NFAT4-cherry are compared. Stimulus (thapsigargin or LTC₄) was applied immediately after resting images were obtained (time zero). At the arrow, stimulus was removed and cells were exposed to Ca²⁺-free solution containing EGTA and cyclosporine A. For the traces labeled N1;Ca²⁺ and N4;Ca²⁺, the stimulus was maintained (controls). Each point is the average of between 14 and 23 cells, taken from 5 and 9 coverslips. Aggregate data are mean ± SEM.

Ca²⁺ Microdomains near Open CRAC Channels Activate NFAT4
(A) Cytoplasmic Ca²⁺ oscillations in response to stimulation of leukotriene receptors with 160 nM LTC₄ in either 2 mM external Ca²⁺ or Ca²⁺-free external solution containing 1 mM La³⁺ are compared.
(B) Graphs summarize data (mean ± SEM) from several experiments. The left graph shows the amplitude of each oscillation plotted against the oscillation (peak) number. The right graph depicts the number of oscillations recorded every 200 s bin after stimulation.
(C) Images compare NFAT4-GFP migration to the nucleus in cells stimulated with LTC₄ either in the presence of external Ca²⁺ or in 0Ca²⁺/La³⁺-containing external solution.
(D) Mean data ± SEM are compared. Each point is the average of between 9 and 14 cells.
(E) Loading the cytoplasm with EGTA (by incubating cells with EGTA-AM) significantly reduced the cytoplasmic Ca²⁺ rise evoked by thapsigargin.
(F) Images compare NFAT4-GFP nuclear accumulation at different times after stimulation with thapsigargin between control cells and a cell exposed to EGTA-AM.
(G) The time course of nuclear NFAT4-GFP is compared between EGTA-loaded cells (open circles) and controls (filled circles) from the same preparations and used on the same days. Each point reflects between 12 and 16 cells. ^∗∗ denotes p < 0.01. Data are represented as mean ± SEM.

Nuclear Ca²⁺ Maintains NFAT4 within the Nucleus
(A) Calcineurin translocates from the cytoplasm to the nucleus after CRAC channel activation. Histone H3 (HH3) was used as a marker for the nuclear fraction and ERK2 was one for the cytoplasmic fraction. Cells were stimulated with thapsigargin for 20 min before lysis. In between the nuclear and cytoplasmic lanes is the marker lane. The histogram summarizes results from three independent experiments. N1, NFAT1; N4, NFAT4; R, resting state; T, thapsigargin stimulation.
(B) Confocal images show the spatial distribution of the parvalbumin constructs used.
(C) Cytoplasmic and nuclear Ca²⁺ measurements are compared. A pulse of Ca²⁺ was applied after store depletion with thapsigargin.
(D) Cytoplasmic and nuclear Ca²⁺ are shown after expression of PV-NLS (untagged).
(E) Images compare NFAT4-GFP accumulation in the nucleus between two control (mock-transfected) cells and one expressing untagged PV-NLS. The thap.+ ionom. image was taken 20 min after ionomycin was applied.
(F) Cytoplasmic and nuclear Ca²⁺ are compared in a cell expressing mutant PV-NLS, which localizes to the nucleus but cannot bind Ca²⁺.
(G) Images show movement of NFAT4-GFP into the nucleus after stimulation of cells expressing mutant PV-NLS.
(H) Cytoplasmic and nuclear Ca²⁺ measurements are shown for a cell expressing PV-NES.
(I) NFAT4-GFP nuclear accumulation is shown for a cell expressing PV-NES.
(J) Aggregate data from several experiments are compared. The white bar on top of the bar for PV-NLS shows the extent of NFAT4-GFP accumulation in response to ionomycin (2 μM) in cells expressing PV-NLS. In these experiments, cells were first stimulated with thapsigargin and NFAT4-GFP accumulation measured (blue bar). Ionomycin was then applied (as in E) and translocation quantified 20 min later. The two bars for PV-NES summarize NFAT4-GFP nuclear levels after 20 and 40 min stimulation with thapsigargin. Data in the histogram reflect mean ± SEM.