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INTRODUCTION |
Physiological engagement of the T cell antigen receptor initiates
a complex series of intracellular signaling events that ultimately
activate the expression of a panel of genes involved in both
initiating and coordinating the immune response (1, 2). The
NFAT1 family of transcription
factors is known to play a pivotal role in the regulation of these
events (3, 4). All members of the NFAT family share a conserved region
of ~270 amino acid residues that is related to the Rel domain and is
involved in sequence-specific DNA binding (5-9). NFAT proteins bind to
DNA as monomers (7), although, in many cases, they have been shown to
bind cooperatively together with a nuclear binding partner, such as
members of the AP-1 family of transcription factors (4, 10-13). NFAT
family members are involved in the transcription of many
immunologically important genes, including the cytokines IL-2, IL-3,
IL-4, IL-5, granulocyte/macrophage colony-simulating factor, and tumor
necrosis factor-
, and several cell-surface molecules such as CD40L
and FasL (3, 4). Three members of the NFAT family are expressed in T
cells: NFATp (NFAT1/NFATc2), NFATc (NFAT2/NFATc1), and NFAT4 (NFATx/NFATc3) (5-9). Analysis of mice deficient in individual NFAT
family members has revealed that these proteins play distinct and
largely non-overlapping functions in T cell biology (14-19). In
addition to their effects in lymphocytes, NFAT family members have also
been shown to play a variety of roles in other non-lymphoid tissues
(20-23).
NFAT proteins appear to be regulated primarily at the level of their
subcellular localization (3, 4, 24-26). They are normally located in
the cytoplasm of resting cells in a latent form, but are induced to
enter the nucleus in response to an elevation in the intracellular
calcium concentration and the subsequent action of the
calcium-regulated serine/threonine phosphatase calcineurin (3, 4,
24-26). Once activated, calcineurin directly dephosphorylates a number
of highly conserved serine residues located in the NFAT N-terminal
regulatory domain, causing NFAT proteins to translocate rapidly into
the nuclear compartment, bind their DNA target sequences, and activate
gene transcription (4, 25, 26). The calcineurin-mediated dephosphorylation and nuclear localization of NFAT are directly counteracted by a number of protein kinases (27-32). These kinases can
either antagonize NFAT nuclear translocation or promote the nuclear
export of NFAT proteins. As a result, efficient initiation of
NFAT-dependent gene expression requires a sustained
increase in the intracellular calcium concentration (33-35),
presumably to maintain calcineurin in an active state capable of
overcoming the inhibitory effects of the NFAT kinases. In addition,
sustained signaling through the calcium/calcineurin pathway is required to maintain NFAT-dependent transcription since the
inhibition of calcineurin is known to attenuate ongoing
NFAT-dependent gene expression (28). Furthermore, the
efficiency of NFAT-dependent gene expression has been shown
to be extremely sensitive to the frequency of intracellular calcium
oscillations (35). Accordingly, NFAT-dependent
transcription is highly dynamic and exquisitely sensitive to both
quantitative and qualitative changes in the calcium/calcineurin
signaling pathway.
Although much attention has been paid to the regulation of NFAT
subcellular localization, less is known about the regulation of the
intrinsic DNA binding activity of NFAT. However, given the dynamic
nature of NFAT-dependent gene expression, it is likely that
the interaction of NFAT with DNA is also tightly regulated. In fact,
previous work has suggested that the DNA binding activity of NFATp is
influenced by its state of phosphorylation, although the mechanisms
involved remain unclear (36-38). In this study, we investigated the
regulation of NFATc DNA binding activity. We demonstrate that the
intrinsic DNA binding activity of NFATc is negatively regulated by
phosphorylation. Furthermore, we identify the three highly conserved
Ser-Pro (SP) repeat motifs within the NFAT homology domain as key
quantitative determinants of NFATc DNA binding affinity and provide
evidence that phosphorylation of these residues by the protein kinase
GSK-3 inhibits the ability of NFATc to bind DNA. Taken together,
our studies afford new insights into the regulation of NFATc and
underscore the potential role of GSK-3 in the regulation of
NFAT-dependent gene expression.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
COS and Jurkat TAg cells
were maintained at 37 °C in 7.5% CO2 in growth medium
(RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10%
(v/v) fetal calf serum (Hyclone Laboratories, Inc.), 100 units/ml
penicillin G, and 100 µg/ml streptomycin). For COS cell
transfections, cells (107) were resuspended in 0.4 ml of
growth medium and placed in an electroporation cuvette (0.4-cm gap;
Bio-Rad) together with the indicated amount of plasmid DNA (3 µg for
NFATc expression vectors and 10 µg for GSK-3
-S9A). The
amount of plasmid DNA was held constant by addition of the pcDNA3
vector control. After incubation at room temperature for 5 min, cells
were exposed to an electric field of 230 V at a capacitance of 960 microfarads (Gene Pulser II, Bio-Rad) and, after a 5-min recovery
period, were replated in growth medium and placed at 37 °C. Jurkat
TAg cell transfections were similar, except that an electric field of
250 V and 10 µg of NFATc expression vector were used.
Expression Constructs and Recombinant Proteins--
The
FLAG-tagged wild-type and mutant NFATc expression constructs in the
mammalian expression vector pBJ5 have been described previously (39).
The NFATc mutants comprise the indicated serine-to-alanine substitutions: NFATc-mSRD (S172A, S175A, S176A, S179A, S181A, S184A, S187A, S188A, S191A, and S194A), NFATc-mSPx1 (S199A, S203A, S207A, and S211A), NFATc-mSPx2 (S199A, S203A, S207A, and
S211A; and S278A, S282A, S286A, and S290A), and NFATc-mSPx3
(S199A, S203A, S207A, and S211A; S233A and S241A; and S278A, S282A,
S286A, and S290A). The NFATc-N
415 deletion mutant encodes NFATc
amino acids 415-718 in the pBJ5 mammalian expression vector. For
in vitro translation constructs, wild-type and mutant NFATc
constructs without the FLAG tag were subcloned into
pCITE-4b+ (Novagen). GSK-3
-S9A in pcDNA3 was a
generous gift of E. G. Krebs (40).
Whole Cell Extracts (WCE) and Electrophoretic Mobility Shift
Assay (EMSA)--
40 h post-transfection, COS cells were treated for
30 min with 3 µM ionomycin (Calbiochem) plus 10 mM CaCl2 in the presence or absence of FK506 (5 ng/ml). Where indicated, 20 mM LiCl was also added to
cells. Cells were washed twice with cold phosphate-buffered saline (137 mM NaCl, 2.5 mM KCl, 10 mM
Na2HPO4, and 2 mM
NaH2PO4, final pH 7.4), scraped off plates, and
briefly centrifuged at room temperature at 15,000 × g.
Cell pellets were vigorously resuspended in 0.15 ml of WCE buffer (20 mM HEPES-KOH, pH 7.6, 25% glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.2 mM
EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 2.5 µg/ml leupeptin) plus phosphatase
inhibitors (50 mM
-glycerophosphate, 50 mM
sodium fluoride, and 0.5 mM sodium orthovanadate) and then
incubated on ice for 20 min. Cell lysates were centrifuged at
18,000 × g for 5 min at 4 °C, and the supernatants were saved. For EMSA, 1.5 µl of WCE was added to a final volume of 20 µl of binding mixture, which contained 5 µg of poly(dI-dC), 10 mM Tris, pH 7.5, 0.5 mM EDTA, 5% glycerol, and
0.3 ng of 32P-labeled NFAT oligonucleotide probe. After
incubation at room temperature for 45 min, the resulting protein-DNA
complexes were resolved by electrophoresis on a 4% native
polyacrylamide gel, dried, and exposed to Kodak XAR film overnight.
Quantitation of the data was performed by scanning densitometry of the
film using a Bio-Rad GS-700 imaging densitometer and Molecular Analyst
Version 1.5 software. The oligonucleotide probes used in EMSA were
5'-ctagTGGTGTAATAAAATTTTCCAATGT-3' (
86 to
63 of the murine IL-4
promoter) and its complement, 5'-ctagACATTGGAAAATTTTATTACACCA-3'.
For the experiments involving the in vitro dephosphorylation
of NFATc, NFATc-transfected cells were harvested in WCE buffer lacking
phosphatase inhibitors. WCE were then incubated in phosphatase buffer
(50 mM Tris-HCl, pH 8.5, and 0.1 mM EDTA) in
either the presence or absence of 1 unit of calf intestinal alkaline
phosphatase (Roche Molecular Biochemicals) at 37 °C for 1 h,
and DNA binding activity was assayed by EMSA. Phosphatase inhibitors
were added where indicated. To measure the effect of endogenous kinases
on NFATc DNA binding activity, WCE prepared from ionomycin-stimulated NFATc-transfected cells were incubated at 37 °C in the presence or
absence of 1 mM ATP for 1 h and analyzed by EMSA.
For the experiment shown in Fig. 4B, NFATc-transfected
Jurkat TAg cells were stimulated with 2 µM ionomycin for
30 min, and COS cells were stimulated as described above. Where
indicated, LiCl was added to a final concentration of 20 mM. Cell extracts were prepared by lysis in cell extract
buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl,
1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM EDTA,
0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 2.5 µg/ml leupeptin, and phosphatase inhibitors).
Immunoblotting and SDS-PAGE--
Cell extracts were mixed with
an equal volume of 2× SDS gel loading buffer (100 mM Tris,
pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, and 2%
2-mercaptoethanol) and heated to 100 °C for 5 min. Samples were
resolved on an 8% SDS-polyacrylamide gel and transferred to
nitrocellulose. The nitrocellulose membrane was incubated with the
anti-FLAG M2 mAb (1 µg/ml; Sigma) for 1 h, washed three times
with cold phosphate-buffered saline, and then incubated with a 1:2000
dilution of horseradish peroxidase-conjugated rabbit anti-mouse IgG
(Zymed Laboratories Inc.) for 1 h. The blots were
washed again and visualized using ECL (Amersham Pharmacia Biotech) and
Kodak XAR film. In vitro translated samples labeled with
[35S]methionine (Amersham Pharmacia Biotech) were
resolved on an 8% SDS-polyacrylamide gel, dried, and exposed to Kodak
XAR film overnight.
In Vitro Translation--
In vitro transcription and
translation of wild-type and mutant NFAT-pCITE-4 constructs were
performed using the Single Tube Protein System 2 (Novagen) as
recommended by the manufacturer. Reactions were supplemented with
either [35S]methionine for SDS-PAGE analysis or unlabeled
methionine for EMSA.
GSK-3 in Vitro Phosphorylation Reactions--
To determine the
effects on NFATc of GSK-3 in vitro phosphorylation, 1.5 µl
of either the wild-type or mutant NFATc in vitro translation
products were incubated at 30 °C for 20 min in kinase buffer (20 mM Tris-Cl, pH 7.5, 10 mM MgCl2,
and 5 mM dithiothreitol) supplemented with 0.5 mM ATP, 1.5 µg of poly(dI-dC), and 5% glycerol in either
the presence or absence of 5 units of purified GSK-3
(New England
Biolabs Inc.). Subsequently, 0.3 ng of radioactive probe was added; the
reaction was incubated at room temperature for 20 min; and protein-DNA
complexes were separated by native gel electrophoresis. To determine
the effects of GSK-3 phosphorylation on preformed NFATc·DNA
complexes, either the wild-type or mutant NFATc in vitro
translation products were incubated in kinase buffer supplemented with
0.5 mM ATP, 1.5 µg of poly(dI-dC), 5% glycerol, and
radiolabeled NFAT probe for 20 min at room temperature to allow for
efficient NFATc-DNA binding. Reactions were then incubated in
either the presence or absence of 5 units of purified GSK-3 at 30 °C
for the indicated times, and protein-DNA complexes were separated by
native gel electrophoresis.
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RESULTS |
NFATc DNA Binding Activity Is Negatively Regulated by
Phosphorylation--
Whereas previous studies on NFATc have largely
focused on the regulation of its subcellular localization, we have
chosen to investigate the regulation of its DNA binding activity. We
first wanted to determine the effects of calcineurin activation on the DNA binding activity of NFATc. COS cells were used as a convenient model system for this study since they are known to support
calcium-dependent, FK506-sensitive changes in NFATc
subcellular localization, yet lack endogenous NFAT proteins (29, 32,
39). Thus, COS cells were transfected with an expression vector
encoding FLAG-tagged wild-type NFATc and stimulated with calcium
ionophore in either the presence or absence of FK506. Whole cell
extracts were subsequently prepared and analyzed by EMSA using a
radiolabeled NFAT probe derived from the murine IL-4 promoter (41). By
using whole cell extracts, we were able to eliminate differences due to
alterations in subcellular localization and instead focus on
differences in the intrinsic DNA binding activity of NFATc. As
expected, no specific NFAT DNA binding was detected in extracts made
from mock-transfected COS cells (Fig. 1,
upper panel, lane 1), even following stimulation with the calcium ionophore ionomycin (data not shown). A low level of
DNA binding was observed in extracts prepared from non-stimulated NFATc-transfected cells, whereas stimulation of NFATc-transfected cells
with ionomycin resulted in a marked increase in NFATc-specific DNA
binding activity (Fig. 1, upper panel, lanes 2 and 3). This ionomycin-induced increase in NFATc DNA binding
activity was potently inhibited by the presence of the
immunosuppressant drug FK506 (Fig. 1, upper panel,
lane 4), indicating a role for calcineurin. The specificity
of the DNA binding complex was confirmed by its competition with an
excess of unlabeled IL-4 probe, but not with an excess of an unrelated
oligonucleotide (data not shown). Importantly, the complex was also not
competed with a consensus AP-1 probe (data not shown). This observation
indicates that the observed gel shift did not result from cooperative
binding of NFATc and AP-1, but rather represents NFATc binding to the
DNA probe alone. Control immunoblot analysis revealed that the levels
of NFATc expression were equivalent in each of the extracts (Fig. 1,
lower panel). As previously reported, ionomycin stimulation
resulted in an increase in the mobility of NFATc on SDS-PAGE, an effect that was inhibited by FK506 (6, 39). Taken together, these results
indicate that ionomycin stimulation acts through calcineurin to
increase the intrinsic DNA binding affinity of NFATc. A similar effect
of calcium ionophore treatment on the DNA binding activity of NFATp has
also been observed (36, 38).

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Fig. 1.
Calcineurin regulates the DNA binding
activity of NFATc. Upper panel, COS cells transiently
transfected with FLAG-tagged wild-type NFATc were left non-stimulated
(NS) or were treated with 3 µM ionomycin
(Iono) or 3 µM ionomycin plus 5 ng/ml FK506
(Iono + FK506) for 30 min. WCE were prepared, and DNA
binding activity was measured by EMSA. The asterisk
indicates a nonspecific band observed in all lanes. The relative
intensities (R.I.) of the specifically shifted complexes
(arrowhead) were determined by scanning densitometry and are
indicated below each lane. Lower panel, parallel samples
were analyzed by 8% SDS-PAGE, followed by immunoblotting with the
anti-FLAG M2 mAb.
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Since it is well established that calcineurin promotes nuclear
translocation of NFATc through dephosphorylation of regulatory serine
residues (4, 25, 26), we next chose to test whether the phosphorylation
status of NFATc plays a direct role in regulating its DNA binding
activity. Accordingly, whole cell extracts prepared from non-stimulated
NFATc-transfected cells were incubated in vitro with calf
intestinal alkaline phosphatase. As shown in Fig. 2A, this treatment resulted in
a dramatic increase in NFATc-dependent DNA binding
activity, an effect that was inhibited by the presence of phosphatase
inhibitors. Thus, it appears that dephosphorylation of NFATc in
vitro is able to directly increase its ability to bind DNA, which
is consistent with previous findings for NFATp (37). Next we performed
the converse experiment by incubating extracts that contained a high
level of NFATc DNA binding activity in the presence of ATP. As shown in
Fig. 2B, NFATc DNA binding activity was greatly diminished
in extracts incubated with ATP compared with control extracts incubated
without ATP. Presumably, the endogenous protein kinases present in the
extracts are able to rephosphorylate NFATc and to inhibit its ability
to bind DNA. These results strongly suggest that the phosphorylation
status of NFATc plays an important role in regulating its ability to bind DNA.

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Fig. 2.
NFATc DNA binding activity is negatively
regulated by phosphorylation. A, WCE prepared from COS
cells transiently transfected (TF) with FLAG-tagged
wild-type NFATc were incubated at 37 °C for 1 h in the presence
of buffer alone (cont), alkaline phosphatase
(+AP), or alkaline phosphatase plus phosphatase inhibitors
(+AP+PI) and analyzed for DNA binding activity by EMSA.
B, WCE prepared from ionomycin (Iono)-stimulated
COS cells expressing FLAG-tagged wild-type NFATc were incubated in the
presence or absence of 1 mM ATP at 37 °C for 1 h
and analyzed for DNA binding activity by EMSA. The relative intensities
(R.I.) of the specifically shifted complexes were determined
by scanning densitometry and are indicated below each lane.
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Identification of the NFATc Ser-Pro Repeats as Critical
Determinants of DNA Binding Activity--
Having implicated the
phosphorylation state of NFATc as an important determinant of its
ability to bind DNA, we next wanted to identify the specific regulatory
amino acid residues that mediate this effect. To identify the region of
NFATc responsible for the phosphorylation-dependent regulation
of DNA binding, we first examined the effects of ionomycin stimulation
on the DNA binding activity of an NFATc deletion mutant (NFATc-N
415)
composed of only the C-terminal Rel homology domain. As shown in Fig.
3B, the C-terminal fragment of
NFATc bound DNA constitutively and was unaffected by ionomycin
stimulation. This constitutive DNA binding activity observed following
removal of the NFATc N-terminal 415 amino acids suggests that the
N-terminal region normally acts to inhibit the activity of the
C-terminal DNA binding domain. Furthermore, the lack of ionomycin
responsiveness suggests that the N-terminal region, not the C-terminal
DNA binding domain, is likely to contain the regulatory phosphoamino
acid residues targeted by calcineurin.

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Fig. 3.
Conserved SP repeat motifs are critical
determinants of NFATc DNA binding activity. A, shown is
a schematic of the FLAG-tagged NFATc mutants used in this study. The
various serine-to-alanine substitutions in the conserved SRD and SP
repeat motifs are indicated. B, shown are the results from
EMSA analysis of WCE prepared from COS cells transiently transfected
(TF) with either control plasmid (mock) or
NFATc-N 415 and either left non-stimulated (NS) or treated
with 3 µM ionomycin (Iono) for 30 min. The
mobility of the specifically shifted complex is indicated by the
arrowhead, and the nonspecific band is indicated by the
asterisk. C, WCE were prepared from COS cells
transiently transfected with plasmids encoding the indicated
FLAG-tagged mutant NFATc molecules and analyzed for DNA binding
activity by EMSA (upper panel). The relative intensities
(R.I.) of the specifically shifted complexes were determined
by scanning densitometry and are indicated below each lane. Parallel
samples were analyzed by 8% SDS-PAGE, followed by immunoblotting with
the anti-FLAG M2 mAb (lower panel).
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The N terminus of NFATc is known to be highly phosphorylated and to
contain several functionally important conserved sequence motifs rich
in serine residues (4, 25, 26), including the serine-rich domain (SRD)
and three Ser-Pro repeat motifs (Fig. 3A). Both of these
sequence motifs have previously been implicated in the
phosphorylation-dependent control of NFATc subcellular localization (29, 39). To investigate the potential role of each of
these motifs in the regulation of phosphorylation-dependent NFATc DNA binding activity, we examined the DNA binding activity of
NFATc mutants containing substitutions of serine to
non-phosphorylatable alanine residues in each of these conserved
sequences. The NFATc-mSPx3 mutant, in which all three SP repeat motifs
were alanine-substituted, was found to constitutively bind DNA even in
the absence of ionomycin stimulation (Fig. 3C, upper
panel, lane 3). In fact, ionomycin stimulation did not
further increase the level of DNA binding of this NFATc mutant (see
Fig. 4B, compare lanes 5 and 6). In contrast, the alanine-substituted NFATc-mSRD mutant, which is known to
be constitutively localized to the nucleus (39), did not exhibit
significant DNA binding activity (Fig. 3C, upper
panel, lane 4). In addition, the NFATc-mSRD-mSPx3
double mutant, which contains alanine substitutions in both the SRD and
the SP repeat motifs, exhibited a similar level of DNA binding activity
to that of the NFATc-mSPx3 mutant (Fig. 3C, upper
panel, lane 5). Together, these results suggest that
the conserved SP repeat motifs, but not the SRD, play an important role
in the regulation of NFATc DNA binding activity. Strikingly, we found a
strong correlation between the number of alanine-substituted SP repeats
and the degree of NFATc- DNA binding. The highest level of DNA binding
was observed for NFATc-mSPx3, with progressively lower levels of
binding observed for NFATc-mSPx2 and NFATc-mSPx1 (Fig. 3C,
upper panel, lanes 1-3). These differences in
DNA binding could not be explained by differences in protein expression
since immunoblot analysis revealed that the NFATc mutants were all
expressed at equivalent levels (Fig. 3C, lower
panel). Interestingly, in addition to their effects on DNA binding
activity, we observed a strong correlation between the number of
alanine-substituted SP repeats and the migration of the NFATc mutants
detected by SDS-PAGE. The NFATc-mSPx3 mutant migrated with the fastest
mobility, whereas NFATc-mSPx2 and NFATc-mSPx1 migrated progressively
more slowly (Fig. 3C, lower panel, lanes 1-3). Notably, each of the alanine-substituted SP repeat mutants migrated more rapidly compared with wild-type NFATc (see Fig. 4C). These differences in migration on SDS-PAGE most likely
represent the effects of differential phosphorylation of the SP repeats and suggest that each of the individual SP repeat motifs in NFATc is
likely to be phosphorylated in these cells. Taken together, these
results identify the conserved SP repeat motifs as key determinants of
NFATc DNA binding activity. Furthermore, it appears that the phosphorylation status of these motifs is likely to play an important role in regulating the degree of NFATc-DNA binding.
Evidence of a Role for GSK-3 in the Regulation of NFATc DNA Binding
Activity in COS Cells--
Previous work has shown that GSK-3 can
phosphorylate the SP repeat motifs of NFATc and is thought to promote
NFATc nuclear export (29). Since the SP repeat motifs also appear to
regulate DNA binding activity, GSK-3 seemed a likely candidate as a
potential regulator of NFATc DNA binding activity. We therefore
investigated whether manipulation of GSK-3 activity in cells could
affect the DNA binding activity of NFATc. Previous work has
demonstrated that LiCl can directly inhibit the enzymatic activity of
GSK-3 in cells (42, 43); therefore, we tested the effect of LiCl treatment on NFATc DNA binding activity. As shown in Fig.
4A (upper panel,
lane 4), treatment of NFATc-transfected COS cells with LiCl
induced NFATc to bind DNA at a level comparable to that of ionomycin
stimulation, whereas treatment of cells with both ionomycin and LiCl
resulted in an additive increase in DNA binding activity. Immunoblot
analysis revealed equivalent levels of protein expression and that
stimulation with both ionomycin and LiCl resulted in a marked increase
in the migration of NFATc as detected by SDS-PAGE (Fig. 4A,
lower panel). This increase in migration of NFATc likely indicates the generation of highly dephosphorylated forms of NFATc, presumably caused by the combined effects of calcineurin-mediated NFATc
dephosphorylation and the attenuation of NFATc rephosphorylation by the
LiCl-mediated inhibition of GSK-3. Taken together, these results are
consistent with the notion that GSK-3-mediated phosphorylation acts to
negatively regulate the DNA binding activity of NFATc.

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Fig. 4.
Effects of LiCl treatment and GSK-3
overexpression on the DNA binding activity of NFATc in transiently
transfected COS cells. A: upper panel, COS
cells transiently transfected with FLAG-tagged wild-type NFATc were
left non-stimulated (NS) or were treated with 3 µM ionomycin (Iono), 20 mM LiCl,
or 3 µM ionomycin plus 20 mM LiCl (Iono + LiCl). WCE were prepared and analyzed for specific DNA binding
activity by EMSA. The relative intensities (R.I.) of the
specifically shifted complexes were determined by scanning densitometry
and are indicated below each lane. Lower panel, parallel
samples were analyzed by 8% SDS-PAGE, followed by immunoblotting with
the anti-FLAG M2 mAb. B: upper panel, COS cells
were cotransfected (TF) with either control plasmid or an
expression vector encoding GSK-3 -S9A together with a plasmid
encoding either FLAG-tagged wild-type NFATc (lanes 1-4) or
the FLAG-tagged NFATc-mSPx3 mutant (lanes 5-8). The cells
were either left non-stimulated or treated with ionomycin for 30 min,
and WCE were prepared and analyzed for DNA binding activity by EMSA.
Lower panel, parallel samples were analyzed by 8% SDS-PAGE,
followed by immunoblotting with the anti-FLAG M2 mAb. C:
upper panel, Jurkat cells (lanes 1-3) or COS
cells (lanes 4-9) were transfected with a plasmid encoding
FLAG-tagged wild-type or mutant NFATc. Cells were either left untreated
or treated with ionomycin or ionomycin plus LiCl. Cell extracts were
prepared and analyzed by 6% SDS-PAGE, followed by immunoblotting with
the anti-FLAG M2 mAb. Lower panel, shown is an extended
exposure of lanes 1-3.
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To further investigate the potential role of GSK-3 in the regulation of
NFATc, we examined the consequences of GSK-3 overexpression on the
ionomycin-induced increase in NFATc DNA binding activity. The GSK-3
protein kinase is normally constitutively active in resting cells, but
is inhibited when phosphorylated by a number of mitogen-activated
protein kinases (44-46). Consequently, to ensure that GSK-3 remained
active in our assays, we used a GSK-3
mutant (GSK-3
-S9A) that
contains an alanine substitution at Ser9, the principal
regulatory site of phosphorylation, and is therefore resistant to
mitogen-induced inhibition (40). As shown in Fig. 4B
(upper panel, compare lanes 2 and 4),
overexpression of GSK-3
-S9A attenuated the increase in NFATc DNA
binding activity following stimulation with ionomycin. Similar results
were obtained with wild-type GSK-3
(data not shown). This effect
appears to be mediated by the conserved SP repeats, as overexpression
of GSK-3
-S9A did not have an effect on the DNA binding activity of
the mutant NFATc-mSPx3 (Fig. 4B, upper
panel, lanes 5-8).
Next we examined how the extent of phosphorylation and
dephosphorylation of NFATc that we have observed in COS cells relates to that in T cells. Thus, we compared the migration of FLAG-tagged NFATc expressed in COS cells with that expressed in the Jurkat T cell
line. As shown in Fig. 4C, NFATc isolated from either COS or
Jurkat cells under non-stimulated conditions exhibited a similar migration on SDS-PAGE, indicating that, under resting conditions, NFATc
is likely to be phosphorylated to a similar extent in the two different
cell types (Fig. 4C, upper panel, compare
lanes 1 and 4). Whereas treatment with ionomycin
resulted in a modest increase in the migration of NFATc in both cell
types, a more marked increase in the migration of NFATc was observed
following stimulation with both ionomycin and LiCl. Initial inspection
of the immunoblot indicated that these increases in NFATc migration were more profound in COS cells than in the Jurkat cell line. However,
it is important to note that the fastest migrating forms of NFATc
observed in ionomycin/LiCl-treated Jurkat cells appeared to comigrate
with the fastest migrating NFATc forms observed in ionomycin/LiCl-treated COS cells. This observation was more evident upon examination of a longer exposure of the Jurkat samples (Fig. 4C, upper panel, compare lanes 3 and
6; and lower panel, lane 3). Thus, it
appears that the absolute degree of NFATc dephosphorylation, as
measured by mobility shift, is similar in both Jurkat and COS cells,
although the efficiency of dephosphorylation in the two cell types may
differ. Moreover, these results indicate that LiCl markedly affects the
migration of NFATc in both cell types, presumably by preventing GSK-3
from phosphorylating NFATc. Finally, it should be noted that the
NFATc-mSPx3 mutant exhibited a similar migration to the most rapidly
migrating forms of NFATc detected in ionomycin/LiCl-treated Jurkat and
COS cells. This observation is consistent with the notion that the
NFATc-mSPx3 mutant does indeed mimic a highly dephosphorylated form of NFATc.
Effects of GSK-3 Treatment on the DNA Binding Activity of NFATc in
Vitro--
Next we wanted to test whether purified GSK-3 could
directly inhibit NFATc DNA binding activity in vitro. For
these experiments, in vitro translated wild-type NFATc was
incubated in either the presence or absence of purified GSK-3
and
then examined for DNA binding activity by EMSA. As shown in Fig.
5A, the ability of the
in vitro translated wild-type NFATc to bind DNA was potently inhibited by incubation with purified GSK-3
. This effect appeared to
be dependent upon the SP repeat motifs, as the NFATc-mSPx3 mutant was
largely resistant to the inhibitory effects of GSK-3
(Fig.
5A, lanes 3 and 4). Concomitant with
its effects on NFATc-DNA binding, GSK-3
treatment had a significant
effect on the mobility of wild-type NFATc as detected by SDS-PAGE (Fig.
5B). This difference in mobility presumably reflects the
GSK-3-mediated phosphorylation of the SP repeat motifs since the
NFATc-mSPx3 mutant was unaffected by GSK-3 treatment. Thus, it appears
that treatment with GSK-3
potently inhibits the DNA binding activity
of NFATc, presumably via phosphorylation of the SP repeat motifs. It
should be noted that, in many cases, the phosphorylation of a protein
substrate by GSK-3 is dependent upon the prior activity of a priming
kinase (47). We believe it is therefore possible that GSK-3 may not act
alone to phosphorylate NFATc, but might require the prior action of a
priming kinase present in the in vitro
transcription/translation extracts used in these experiments.

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Fig. 5.
Treatment with GSK-3
inhibits the DNA binding activity of NFATc in
vitro. A, in vitro
translated (IVT) wild-type NFATc or NFATc-mSPx3 was
incubated in the presence or absence of purified GSK-3 at 30 °C
for 20 min and DNA binding activity was analyzed by EMSA. B,
35S-labeled in vitro translated wild-type NFATc
or NFATc-mSPx3 was incubated in the presence or absence of purified
GSK-3 for 20 min at 30 °C and analyzed by 8% SDS-PAGE, followed
by autoradiography. C-E, in vitro translated
wild-type NFATc (C and D) or NFATc-mSPx3
(E) was allowed to form a complex with a
32P-labeled DNA probe at room temperature. Samples were
then incubated at 30 °C for the indicated time periods with either
GSK-3 (C and E) or buffer alone
(D), and the level of NFATc remaining bound to the probe was
assessed by EMSA. The relative intensities (R.I.) of the
specifically shifted complexes were determined by scanning densitometry
and are indicated below each lane.
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Finally, we wanted to test whether treatment with GSK-3
could affect
preformed NFATc·DNA complexes. Hence, in vitro translated NFATc was first incubated with radiolabeled probe and allowed to form
NFATc·DNA complexes. The complexes were then incubated at 30 °C in
the presence or absence of purified GSK-3
for various periods of
time and analyzed by EMSA. As shown in Fig. 5 (C and D), treatment with GSK-3
promoted a rapid reduction in
the level of DNA-bound wild-type NFATc compared with mock-treated
samples. In contrast, GSK-3
treatment had little effect on the DNA
binding activity of the NFATc-mSPx3 mutant (Fig. 5E). Thus,
GSK-3 treatment leads to a rapid reduction in the level of NFATc DNA
binding activity, an effect that is dependent upon the integrity of the
SP repeat motifs. Presumably, GSK-3 is able to shift the equilibrium in favor of unbound NFATc, either by phosphorylating DNA-bound NFATc and
inducing its dissociation or by phosphorylating dissociated NFATc and
precluding its reassociation with DNA.
 |
DISCUSSION |
In this study, we provide evidence that the intrinsic DNA binding
activity of NFATc is negatively regulated by phosphorylation. We show
that dephosphorylation of NFATc in vitro and activation of
calcineurin in cells lead to enhanced NFATc- DNA binding, whereas phosphorylation of NFATc in vitro potently inhibits its
ability to bind DNA. We believe that this phosphorylation-sensitive
regulation of DNA binding is likely to be a shared general property of
the NFAT family since both the in vitro and in
vivo dephosphorylation of NFATp have also previously been shown to
enhance its DNA binding activity (36-38). Importantly in this study,
we identify the conserved SP repeat motifs as critical quantitative
determinants of NFATc DNA binding activity. Finally, we demonstrate
that the GSK-3 protein kinase can act to inhibit NFATc DNA binding
activity, an effect that appears to be mediated by phosphorylation of
the SP repeats. Taken together, these studies afford new insights into
the regulation of NFATc and highlight a potential role for the GSK-3
protein kinase in the regulation of NFATc-dependent gene expression.
One of the principal findings of our study is that the phosphorylation
of the conserved SP repeat motifs appears to play an important role in
negatively regulating the DNA binding activity of NFATc. In support of
this notion, we have shown that alanine substitution of the conserved
serine residues in these motifs leads to a marked increase in the level
of NFATc DNA binding activity. In addition, we have shown that the SP
repeat motifs are critically required for the inhibitory effects of the
GSK-3 protein kinase on NFATc-DNA binding. Furthermore, through the
analysis of NFATc mutants with increasing numbers of
alanine-substituted SP repeats, we have provided evidence that the
state of phosphorylation of the SP repeats motifs is inversely related
to the degree of NFATc-DNA binding. This latter observation suggests
that the phosphorylation state of these motifs does not appear to act
as a simple binary switch to regulate NFATc-DNA binding, but rather
implies that the differential phosphorylation of the SP repeats might
act to quantitatively regulate the DNA binding activity of NFATc. Thus, we propose that the phosphorylation status of the NFATc Ser-Pro repeat
motifs is likely to act as a form of molecular rheostat by integrating
the relative activities of both the calcium/calcineurin signaling
pathway and the opposing NFATc kinase(s), thereby determining the
ultimate degree of NFATc DNA binding activity.
Our identification of the conserved SP repeat motifs as critical
quantitative determinants of NFATc DNA binding activity suggests that
the kinases that directly phosphorylate these residues are likely to
have an important influence on the regulation of
NFATc-dependent gene expression. In particular, by opposing
the action of calcineurin and acting to attenuate the DNA binding
activity of NFATc, they are likely to make an important contribution to
the exquisite calcium sensitivity and dynamic regulation of
NFATc-dependent gene expression observed in vivo
(25, 28, 33-35). Identification of the kinases involved in the
regulation of NFATc DNA binding activity is therefore of considerable
interest. In fact, our results provide several independent lines of
evidence to suggest a potential role for GSK-3 in the
phosphorylation-dependent regulation of NFATc DNA binding
activity. First, LiCl, which is known to be an inhibitor of GSK-3
activity, acted together with ionomycin to enhance NFATc DNA binding
activity in transiently transfected COS cells. Second, overexpression
of a GSK-3 mutant (GSK-3
-S9A) attenuated the ionomycin-induced DNA
binding activity of wild-type NFATc, but had no effect on the
NFATc-mSPx3 mutant. Third, phosphorylation of wild-type NFATc in
vitro with purified GSK-3
strongly inhibited NFATc DNA binding
activity, an effect that appeared to be mediated via phosphorylation of
the conserved SP repeat motifs. Although our studies have implicated a
potential role for GSK-3 in the regulation of NFATc DNA binding
activity, it is important to note that we cannot rule out the
possibility that other cellular protein kinases may be involved.
Interestingly, in addition to the effects of GSK-3 on NFATc DNA binding
activity that we have described here, several other independent studies
also support a potential role for GSK-3 in the regulation of the NFAT
signaling pathway. First, Crabtree and co-workers (29, 48) have
proposed a role for GSK-3 in the regulation of NFATc nuclear export. In
those studies, GSK-3 was identified by biochemical purification and
immunodepletion experiments as the principal cellular kinase
responsible for the phosphorylation of the NFATc Ser-Pro repeat motifs.
Furthermore, LiCl was shown to antagonize the nuclear export of NFATc,
whereas overexpression of GSK-3 was found to enhance NFATc nuclear
export and to inhibit the T cell activation-dependent
induction of an NFAT reporter gene. Second, Ohashi and co-workers (49)
have recently shown that the retroviral expression of the GSK-3
-S9A mutant in murine primary T cells strongly inhibits antigen-induced IL-2
production and T cell proliferation, whereas inhibition of GSK-3
activity with LiCl enhances T cell responses. Third, overexpression of
the Akt/protein kinase B protein kinase, which is known to negatively regulate the activity of GSK-3, has recently been shown to
synergize with Fc
-receptor I stimulation to enhance both NFAT activity and NFAT-dependent cytokine gene expression in
murine bone marrow-derived mast cells (50). Based upon these collective observations, it is tempting to speculate that GSK-3 may play an
important role in the regulation of NFATc-dependent gene
expression. However, the formal demonstration of a role for GSK-3
in
the regulation of NFATc activity in vivo will require either
the generation of more specific GSK-3 inhibitors or the analysis of
cells rendered genetically deficient in GSK-3 protein kinase activity.
Overall, our results suggest that the DNA binding activity of NFATc is
regulated in a phosphorylation-dependent manner by the
differential phosphorylation of the conserved SP repeat motifs. What is
the underlying molecular mechanism that accounts for this observation?
Substantial analysis over the years has indicated that phosphorylation
can influence the DNA binding activity of transcription factors by two
major mechanisms (51). First, phosphorylation of residues within or
close to the DNA binding site itself can directly affect DNA binding
(52-55). Second, phosphorylation can lead to a conformational change
resulting in an indirect modulation of DNA binding activity (56-58).
In the case of NFATc, we have identified the conserved SP repeat motifs
as key phosphorylation-sensitive determinants of NFATc DNA binding
activity. Since these motifs are located in the NFATc N terminus and
are geographically distinct from the C-terminal NFATc DNA binding
domain, a simple model in which phosphorylation directly inhibits DNA
binding appears to be excluded. Rather, our data support an indirect
model of regulation. We have demonstrated that either the removal of
the entire NFATc N terminus or alanine substitution of the conserved SP
repeat motifs results in constitutive DNA binding activity that is
unaffected by the action of calcineurin. This suggests that the
phosphorylated NFATc N terminus is likely to exert an intramolecular
inhibitory influence on the activity of the NFATc C-terminal DNA
binding domain.
There are several potential mechanisms that could explain this negative
regulatory influence. First, the phosphorylated N terminus could engage
in a direct intramolecular interaction with the C-terminal DNA binding
domain and simply occlude its interaction with DNA. In fact,
biochemical evidence supporting a direct intramolecular interaction
between the NFATc N- and C-terminal domains has been provided (39).
This interaction appears to be mediated, at least in part, by the
interaction of phosphorylated serine residues in the SRD with basic
amino acid residues located in the C-terminal nuclear localization
sequence (NLS; 682KRKR685). It has been
proposed that the phosphorylation status of the SRD regulates the
accessibility of the NLS, thereby controlling NFATc nuclear import
(39). In addition to the role of the NLS in nuclear targeting, the
x-ray crystal structure of NFATp has revealed that the NLS is also
directly involved in DNA binding (59). If the accessibility of the NLS
is indeed controlled by the phosphorylation status of the SRD, it might
have been predicted that the NFATc-mSRD mutant would have exhibited
enhanced DNA binding activity, contrary to our findings presented in
Fig. 3B. However, the NLS forms only one of 14 DNA contacts
(59, 60) and does not appear to be absolutely required for DNA binding
per se since recombinant protein fragments of both NFATc and
NFATp that lack the NLS still bind DNA (61, 62). In fact, the other
NFATc DNA contact residues are located distally from the NLS in the N-terminal portion of the Rel homology domain between amino acids 433 and 590. Presumably, it is these latter DNA contact residues that are
precluded from interacting with DNA by the phosphorylated SP repeat
motifs, although whether this inhibition involves a direct interaction
with the phosphorylated SP repeats remains to be seen. A second related
potential mechanism to explain the negative regulatory influence
of the N terminus on the DNA binding activity of NFATc is that the
phosphorylation of the SP repeat motifs could allosterically favor an
NFATc conformation that is incompatible with DNA binding. In fact, such
a mechanism has recently been described for the
phosphorylation-dependent regulation of Ets-1 DNA binding
activity (57). A final possibility is that the phosphorylated N
terminus may bind a specific partner protein that acts to sterically
hinder the association of NFATc with DNA. If that is the case, such a
partner protein would have to be relatively ubiquitous since the
phosphorylation-sensitive regulation of NFATc DNA binding activity was
observed in both COS cells and reticulocyte lysate in vitro
translation extracts. Further insight into the precise molecular
mechanisms involved in the phosphorylation-sensitive regulation of
NFATc DNA binding activity will have to await the detailed structural
analysis of the differentially phosphorylated forms of the full-length
NFATc molecule.