Figure 5.
NFAT4 Is Rephosphorylated within the Nucleus Considerably More Quickly Than NFAT1
(A) Images compare nuclear export of NFAT4-GFP between a control cell and one exposed to leptomycin B (LMB; 100 nM). Nuclear export was initiated by removal of external Ca2+ in the presence of cyclosporine A.
(B) Aggregate data from several experiments are compared.
(C) Western blot shows gel shift of NFAT4-GFP from nuclear extract after stimulation and then at different times (2–20 min) after initiation of export (see bottom of G for timings).
(D) Graph plots relative gel shift as a function of time up to 20 min after the initiation of nuclear export. A decreased gel shift denotes rephosphorylation.
(E) Gel shift for nuclear NFAT1-GFP under conditions identical to (C).
(F) Graph shows relative gel shift for NFAT1-GFP, as in (D).
(G) Gel shift for the SP3NFAT1-NFAT4-GFP construct.
(H) Graph plots relative gel shift for SP3NFAT1-NFAT4-GFP, as in (D). For the gels in (C), (E), and (G), conditions for each lane are shown only in (G). For the resting condition (indicated as Rest), no detectable NFAT was seen in any of the gels because the gels show nuclear extract and, at rest, there is very little NFAT in the nucleus.
(I) Blot compares kinetics of phosphorylation of serine 165 in NFAT4 from the nuclear fraction, denoted as P-S165.
(J) As in (I), but phosphorylation of serine 177 (P-S177) in NFAT1 was measured.
(K) Graphs plot the kinetics of phosphorylation of serine 165 NFAT4 and SP3N1-N4 or serine 177 in NFAT1.
In (C), (E), (G), (I), and (J), HH3 (histone H3) was used as a nuclear marker and ERK was taken as a cytoplasmic marker. Rest denotes the non-stimulated state. Thap + LMB represents the gel shift seen 40 min after stimulation with thapsigargin in the presence of LMB and external Ca2+, when NFAT had fully accumulated in the nucleus. Nuclear extract was isolated at the different times indicated. In all panels, 100 nM leptomycin B was added 10 min before thapsigargin and then maintained throughout. In the graphs, data are represented as mean ± SEM. See also Figure S5.