NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation - PubMed (original) (raw)

NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation

Taichiro Tomida et al. EMBO J. 2003.

Abstract

Transcription by the nuclear factor of activated T cells (NFAT) is regulated by the frequency of Ca(2+) oscillation. However, why and how Ca(2+) oscillation regulates NFAT activity remain elusive. NFAT is dephosphorylated by Ca(2+)-dependent phosphatase calcineurin and translocates from the cytoplasm to the nucleus to initiate transcription. We analyzed the kinetics of dephosphorylation and translocation of NFAT. We show that Ca(2+)-dependent dephosphorylation proceeds rapidly, while the rephosphorylation and nuclear transport of NFAT proceed slowly. Therefore, after brief Ca(2+) stimulation, dephosphorylated NFAT has a lifetime of several minutes in the cytoplasm. Thus, Ca(2+) oscillation induces a build-up of dephosphorylated NFAT in the cytoplasm, allowing effective nuclear translocation, provided that the oscillation interval is shorter than the lifetime of dephosphorylated NFAT. We also show that Ca(2+) oscillation is more cost-effective in inducing the translocation of NFAT than continuous Ca(2+) signaling. Thus, the lifetime of dephosphorylated NFAT functions as a working memory of Ca(2+) signals and enables the control of NFAT nuclear translocation by the frequency of Ca(2+) oscillation at a reduced cost of Ca(2+) signaling.

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Figures

Fig. 1. Imaging of Ca2+-dependent nuclear translocation of GFP–NFAT. (A) Time-lapse images of GFP–NFAT and [Ca2+]i at various time points during and after Ca2+ stimulation as indicated in (B). (B) Changes in GFP fluorescence intensity of the cell shown in (A) in the cytoplasmic region (indicated as Cyt) and the nuclear region (Nuc) are plotted with the change in [Ca2+]i. (C and D) Time courses of GFP–NFAT translocation and [Ca2+]i upon Ca2+ stimulation for 1.5 min [(C), n = 10] or 20 min [(D), n = 8] (mean ± SEM). The ranges of errors are shown only at selected time points for clarity. (E) Dependence of the peak extent of GFP–NFAT nuclear translocation on the duration of Ca2+ stimulation (mean ± SEM; n = 8–21). (F) Efficiency of GFP–NFAT nuclear translocation. Peak nuclear translocation of GFP–NFAT per unit duration of Ca2+ stimulation was plotted against the duration of Ca2+ stimulation.

Fig. 2. Analysis of dephosphorylation of GFP–NFAT. (A) Dephosphorylation and nuclear translocation of GFP–NFAT upon Ca2+ stimulation. The phosphorylation level of GFP–NFAT was analyzed by western blotting with the anti-GFP antibody after the rapid fractionation of BHK cells during Ca2+ stimulation. C, cytoplasmic fraction; N, nuclear fraction. Representative results of three experiments. (B and C) Time-course of the densitometric signals of cytoplasmic-dephosphorylated and nuclear GFP–NFAT (B) and cytoplasmic-phosphorylated GFP–NFAT (C) normalized by total density. (D) Decay of dephosphorylated GFP–NFAT in the cytoplasm after the termination of Ca2+ signaling. Cells were first stimulated with Ca2+ for 3 min, then fractionated at the indicated time-points after Ca2+ removal. Representative results of three experiments are shown. (E) Time-course of the densitometric signal of cytoplasmic dephosphorylated GFP–NFAT normalized by total density.

Fig. 3. Model of Ca2+-dependent nuclear translocation of NFAT. (A) Schematic illustration of a model of NFAT translocation. NFAT assumes one of the three states, cytoplasmic phosphorylated, cytoplasmic dephosphorylated or nuclear transported. Rate constants are defined as indicated. The dephosphorylation rate constant (_k_1) was assumed to be regulated by [Ca2+]i. (B) Model fitting (open circles) to the nuclear translocation time-course of NFAT (continuous trace). Model prediction of the time-course of cytoplasmic-dephosphorylated NFAT is also shown (filled circles). Model para meters used: _k_1, 0.359/min (during Ca2+ stimulation); _k_2, 0.147/min; _k_3, 0.060/min; and _k_4, 0.035/min. (C) Model prediction of NFAT translocation during Ca2+ oscillation. High-frequency Ca2+ oscillation maintains dephosphorylated NFAT in the cytoplasm and induces significant nuclear translocation. Low-frequency oscillation results in intermittent dephosphorylation and induces only slight nuclear translocation of NFAT. Model parameters were the same as those used in (B).

Fig. 4. Ca2+ oscillation frequency dependence of GFP–NFAT nuclear translocation. (A) [Ca2+]i in GFP–NFAT expressing BHK cells challenged with Ca2+ pulses of 0.5 min duration at different intervals (1.5–15 min). (B) GFP–NFAT translocation during oscillatory Ca2+ stimulation. Increase in GFP fluorescence intensity of the nuclear region normalized by that of the entire cell region. (C) Steady-state nuclear translocation of GFP–NFAT is plotted against Ca2+ oscillation interval. Filled circles, experimental results; open circles, model predictions. (D) Dependence of nuclear translocation effectiveness and efficiency on the duty ratio of Ca2+ oscillation. Filled circles, steady-state nuclear translocation level; open squares, efficiency of Ca2+ signals expressed as nuclear translocation divided by duty ratio. (E) Nuclear translocation of GFP–NFAT during Ca2+ stimulation of the same total duration but different patterns. Ca2+ stimulation was applied as either a single continuous 6 min pulse or 12 pulses of 0.5 min duration at intervals of 1.5, 3 or 6 min. The mean ± SEM (n = 5–10) is shown for all results. The ranges of errors are shown only at selected time points for clarity in (A), (B) and (E).

Fig. 5. Activation kinetics of endogenous NFAT in Jurkat T cells. (A) Time-lapse images of GFP–NFAT translocation (upper panel) and samples of anti-NFAT4 immunofluorescence images (lower panel) obtained at the indicated time-points after Ca2+ stimulation. (B) Time courses of nuclear translocation of endogenous NFAT4 (circles) and GFP–NFAT (lines) upon Ca2+ stimulation. Experiments were carried out at room temperature (open circles, n = 9–12; continuous line, n = 7) and 37°C (filled circles, n = 11–17; dashed line, n = 6) (mean ± SEM). The ranges of errors are shown only at selected time points for clarity in the GFP–NFAT experiments. (C) Dephosphorylation of endogenous NFAT4 following Ca2+ stimulation. Whole-cell lysate prepared from Jurkat cells stimulated with Ca2+ at either room temperature or 37°C were used for western blotting with rabbit antiserum raised against NFAT4. Dephosphorylation of NFAT4 resulted in the mobility shift of the immunoblot bands.

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NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation - PubMed (original) (raw)