(Received for publication, December 14, 1995; and in revised form, February 7, 1996)
From the
NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K. T.-Y., Ho, A. M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P. G.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.
NFAT1 (previously termed NFATp) (1, 2, 3) is a member of the NFAT family of
transcription factors that play a key role in the regulation of
cytokine gene transcription during the immune
response(4, 5, 6) . Other members of the NFAT
family include NFATc(7) , NFATx (also known as NFAT4 or
NFATc3)(8, 9, 10) , and NFAT3(9) .
NFAT family proteins are highly homologous in their DNA-binding
domains, which show a weak similarity to the DNA-binding domains of Rel
family proteins(9, 11, 12) . They also
contain a second region (300 amino acids) of moderate sequence
conservation, located immediately N-terminal to the DNA-binding domain,
that we have termed the NFAT homology region. (
)The NFAT
homology region contains several serine- and proline-rich sequence
motifs whose presence and location within the protein are conserved in
all four NFAT family
proteins(8, 9, 10) .
A central
feature of the regulation of NFAT family proteins was inferred from
their pronounced sensitivity to the immunosuppressive drugs cyclosporin
A (CsA) ()and FK506. These drugs (which are potent
inhibitors of cytokine gene transcription in activated T cells, B
cells, mast cells, and natural killer cells) inhibit NFAT-dependent
reporter gene transcription as well as the nuclear appearance of NFAT
DNA binding
activity(1, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
CsA and FK506 act by binding to intracellular immunophilin receptors
and inhibiting the activity of the calmodulin-dependent phosphatase
calcineurin(24, 25) . Overexpression of calcineurin
replaces the calcium requirement for NFAT-dependent transactivation in
T cells and renders the transactivation less sensitive to CsA and
FK506(15, 26, 27) . These results prompted
the suggestion that calcineurin plays a major role in inducible gene
transcription during the immune response, in part by preventing the
nuclear translocation of NFAT family
proteins(28, 29) .
Detailed analysis of the
pre-existing family member, NFAT1, has provided more insight into the
molecular mechanisms by which calcineurin regulates NFAT
activity(30, 31) . ()NFAT1 is present in
the cytoplasm of resting T cells and translocates into the nucleus in
response to stimulation with ionomycin, antigen, or anti-CD3. The
nuclear translocation is preceded by a rapid dephosphorylation that
correlates with a significant increase in the DNA binding affinity of
NFAT1. Each of these three hallmarks of activation (dephosphorylation,
nuclear translocation, and increase in affinity for DNA) is elicited by
calcineurin-dependent mechanisms since each is blocked by CsA and
FK506(30, 31) .
NFAT1 is also effectively
activated by stimulation through the T cell receptor in that it is
rapidly dephosphorylated, it translocates to the nucleus, and it shows
an increased ability to bind DNA. However, this phase of activation is
followed by a return of the bulk of the NFAT1 to its resting state, a
process that correlates with the decline in intracellular free calcium
levels due to feedback mechanisms triggered by the T cell
receptor.
Furthermore, treatment of stimulated T cells with
CsA results in a loss of occupancy of the NFAT sites within the murine
interleukin-2 promoter, as assessed by in vivo footprinting
studies(33) . These observations prompted us to investigate
whether the activation of NFAT1 is dependent on the sustained activity
of calcineurin.
Here we demonstrate that the activation state of NFAT1 is indeed critically dependent on calcineurin activity. We further show a direct interaction between calcineurin and NFAT1 that may underlie the responsiveness of NFAT1 to the level of calcineurin activity in T cells. The interaction is not influenced by the activation state of NFAT1 and is not inhibited by immunosuppressive concentrations of CsA and FK506. The specificity of signaling pathways is often conferred by direct interactions of signaling enzymes with their substrates or with specific targeting proteins within the cell(34, 35, 36, 37, 38, 39, 40) . The NFAT1-calcineurin interaction, which provides a dramatic illustration of this mechanism, may be used to identify new classes of immunosuppressive drugs that block this interaction without inhibiting the enzymatic activity of calcineurin.
Figure 5:
The
N-terminal 415 amino acids of NFAT1 are sufficient to bind calcineurin. A, COS cells were transfected with plasmids encoding
full-length NFAT1 (FL) or the N-terminal 1-415 amino
acids of NFAT1 (1-145). The cells were harvested 48 h
after transfection and lysed in 1% Triton X-100 buffer. The lysates
were incubated with CaM-Sepharose for 1 h at 4 °C. The beads were
washed, boiled in Laemmli reducing buffer, and analyzed by 10% SDS-PAGE
followed by Western blotting with a mixture of anti-NFAT1 and
anti-calcineurin antibodies. Cn, calcineurin. B, the
GST-NFAT1-(1-415) fusion protein was eluted from
glutathione-Sepharose beads and diluted with 3 volumes of 1% Triton
X-100 buffer supplemented with 1 mM CaCl or 5
mM EGTA as indicated, and 100 µl of this mixture was added
to 50 µl of washed, packed CaM-Sepharose beads. In lanes 1 and 3, purified calcineurin (0.3 µg) was added. All
samples were rocked for 1 h at 4 °C and then washed, boiled in
Laemmli reducing buffer, and analyzed by 10% SDS-PAGE followed by
Western blotting with anti-NFAT1 antibody. C, the
GST-NFAT1-(1-415) fusion protein was eluted from
glutathione-Sepharose beads and diluted with 1.5 volumes of 1% Triton
X-100 supplemented with 1 mM CaCl
(lanes
1-7) or 5 mM EGTA (lane 8), and 100 µl
of this mixture was added to 50 µl of washed, packed CaM-Sepharose
beads. Purified calcineurin (0.3 µg), preincubated alone or in the
presence of a 10-fold molar excess of CsA, cyclophilin A (Cyp
A), FK506, FKBP12, or the appropriate drug-immunophilin complex as
indicated, was added to the samples. Following incubation at 4 °C
for 1 h, the CaM-Sepharose beads were washed, boiled in Laemmli
reducing buffer, and analyzed by 10% SDS-PAGE followed by Western
blotting with anti-NFAT1 antibody.
Figure 1: The nuclear localization of NFAT1 is reversed by cyclosporin A. Ar-5 T cells were left unstimulated (A) or were stimulated with 1 µM ionomycin (IONO) for 15 min (B); stimulated with 1 µM ionomycin for 15 min, followed by addition of 1 µM CsA and incubation at 37 °C for a further 30 min (C); or incubated in 1 µM CsA for 15 min prior to stimulation with 1 µM ionomycin for 30 min (D).
The activation of
NFAT1 is also reflected by an increase in its ability to bind to
DNA(30) . We investigated the calcineurin
sensitivity of this effect by testing the DNA binding activity of whole
cell extracts from Ar-5 T cells treated with ionomycin and/or CsA (Fig. 2). Western analysis indicated that each of the extracts
contained NFAT1 (Fig. 2A). The DNA binding activity of
NFAT1 from ionomycin-stimulated cells showed a significant increase
over the DNA binding activity of NFAT1 from unstimulated or CsA-treated
cells (Fig. 2B, compare lanes 7-9 with lanes 1-3 and 4-6). Prior treatment with
CsA prevented the ionomycin-induced increase in the DNA binding
activity of NFAT1 (data not shown). Likewise, treatment with CsA after
ionomycin stimulation returned the DNA binding activity of NFAT1 to a
resting level (compare lanes 10-12 with lanes
1-3), again suggesting that the DNA binding activity of
NFAT1 is continuously regulated by the level of calcineurin activity.
Figure 2: The ionomycin-induced increase in NFAT1 DNA binding activity is reversed by cyclosporin A. A, Ar-5 T cells were left unstimulated (lanes 1 and 2) or were treated for 15 min with 1 µM CsA (lanes 3 and 4); stimulated with 1 µM ionomycin (IONO) for 15 min (lanes 5 and 6); or stimulated with 1 µM ionomycin for 15 min, followed by addition of 1 µM CsA and a further 30-min incubation at 37 °C (lanes 7 and 8). Whole cell extracts were analyzed for NFAT1 content by 6% SDS-PAGE and Western blotting with anti-NFAT1 antibody. The total protein loaded in each lane is indicated at the top. NFAT1 present in extracts from cells treated with CsA following ionomycin stimulation (lanes 7 and 8) was rephosphorylated and comigrated with the bands present in unstimulated and CsA-treated cells (lanes 1-4). B, the whole cell extracts were analyzed for NFAT1 DNA binding activity by electrophoretic mobility shift assay. The arrow indicates the specific NFAT1-DNA complex, so defined as addition of an antibody specific for NFAT1 in the binding reactions completely supershifted the complex (data not shown). The band migrating below the specific NFAT1-DNA complex is nonspecific. The total protein used in the binding reactions is indicated.
The nuclear translocation and increased DNA binding activity of NFAT1 are preceded by a rapid dephosphorylation of the protein (30) that is apparent as an increase in its electrophoretic mobility on SDS gels (Fig. 3A, lanes 1 and 2) and is prevented by preincubation of the cells with CsA (lane 7). CsA reversed the mobility shift within 15 min when added to ionomycin-stimulated cells (lanes 8-10). This effect was observed even in cells pretreated with protein synthesis inhibitors (data not shown), indicating that the reversal was not due to degradation of the modified NFAT1 and its replacement by newly synthesized protein, but rather reflected the rephosphorylation of previously dephosphorylated NFAT1.
Figure 3:
The activation-dependent dephosphorylation
of NFAT1 is reversed by removal of ionomycin, addition of EGTA, or
treatment with CsA. A, addition of CsA or EGTA. Ar-5 T cells
were left unstimulated (lane 1) or were stimulated with 1
µM ionomycin (IONO) for 5 min at 37 °C (lanes 2, 4-6, and 8-10). EGTA
was added to a final concentration of 2 mM, and the cells were
incubated at 37 °C for a further 1, 5, or 15 min (lanes
4-6, respectively). CsA was added to a final concentration
of 1 µM, and the cells were incubated at 37 °C for a
further 1, 5, or 15 min (lanes 8-10, respectively). In lanes 3 and 7, cells were pretreated with 2 mM EGTA or 1 µM CsA, respectively, for 15 min, followed
by stimulation with 1 µM ionomycin for 5 min at 37 °C. B, tryptic phosphopeptide mapping of NFAT1. Ar-5 T cells were
labeled with [P]orthophosphate as detailed under
``Materials and Methods'' and then left unstimulated (left panel) or stimulated with 1 µM ionomycin
for 5 min (middle panel) or stimulated for 5 min with 1
µM ionomycin, followed by a further 30-min incubation in
the presence of 1 µM CsA (right panel). Lysates
from these cells were subjected to anti-NFAT1 immunoprecipitation.
NFAT1 was separated on a 6% SDS-polyacrylamide gel, eluted from the
gel, and digested with trypsin. The samples were electrophoresed in pH
1.9 buffer in the first dimension, followed by chromatography in the
second dimension. Tryptic phosphopeptides were visualized by
autoradiography. Arrowheads indicate the two major tryptic
phosphopeptides that are stimulation-sensitive. C, removal of
ionomycin. Ar-5 T cells were exposed to one cycle of stimulation with 1
µM ionomycin (iono) for 2 min at 37 °C (lane 3), followed by washing and incubation in fresh medium
without ionomycin for 15 min at 37 °C (lane 4). Lanes
5-10 show cells exposed to subsequent cycles of ionomycin
stimulation (2 min), washing, and incubation (15 min) in fresh medium
lacking ionomycin. Lanes 1 and 2 show NFAT1 in Ar-5 T
cells that were left unstimulated or were treated with ionomycin for
the entire period of the experiment, respectively. D, kinetics
of rephosphorylation after removal of ionomycin. Ar-5 T cells were left
unstimulated (lane 1) or were stimulated with 1 µM ionomycin (IONO) for 1 min (lanes 2-5) or
15 min (lanes 6-9) at 37 °C. The stimulated cells (lanes 3-5 and 7-9) were centrifuged
briefly in a microcentrifuge, resuspended in fresh medium without
ionomycin (Wash), and incubated at 37 °C for 1, 5, or 15
min.
The rephosphorylation of NFAT1 mediated by addition of CsA to ionomycin-stimulated cells was confirmed by tryptic mapping of phosphopeptides (Fig. 3B). Two major labeled tryptic peptides (arrowheads) that were observed in NFAT1 immunoprecipitated from unstimulated cells (left panel) were absent in NFAT1 from ionomycin-stimulated cells (middle panel), consistent with previous reports that NFAT1 is partially dephosphorylated in response to ionomycin (30, 31) and suggesting that only certain sites within NFAT1 are dephosphorylated. As expected, this selective dephosphorylation was inhibited by treatment of cells with CsA prior to ionomycin stimulation (data not shown). Treatment of ionomycin-stimulated cells with CsA resulted in the reappearance of the two major labeled peptides (arrowheads in right panel), indicating that following addition of CsA, NFAT1 is rephosphorylated on the same sites that had been dephosphorylated in response to ionomycin.
NFAT1 that had reverted to its original
electrophoretic mobility could be repeatedly dephosphorylated and
rephosphorylated (Fig. 3C). For this experiment, we
took advantage of the reversibility of ionomycin action. Ionomycin
induces the immediate release of calcium stores and stimulates
capacitative calcium entry; conversely, removal of ionomycin allows the
replenishment of calcium stores, thereby deactivating capacitative
calcium entry and returning [Ca]
to resting levels within a few minutes (47, 48) . NFAT1 that had been dephosphorylated during
a 2-min stimulation of Ar-5 T cells with ionomycin (Fig. 3C, lane 3) reverted to the
phosphorylated form upon washing away the ionomycin and incubating the
cells in fresh medium (lane 4). Upon readdition of ionomycin,
the rephosphorylated NFAT1 was again dephosphorylated (lane
5). This successive rephosphorylation and dephosphorylation was
observed for up to five successive cycles of washing the cells and
restimulating them with ionomycin (lanes 3-10) (data not
shown), again indicating that rephosphorylation returns NFAT1 to a
state similar (if not identical) to that found in resting cells.
We
examined the kinetics of NFAT1 rephosphorylation in Ar-5 cells
stimulated with ionomycin for 1 min, at which time NFAT1 has not yet
translocated to the nucleus, or for 15 min, by which time NFAT1 is in
the nucleus of all cells(30) . Removal of ionomycin by washing
resulted in both cases in the rapid rephosphorylation of NFAT1 (Fig. 3D, lanes 3-5 and 7-9). The simplest explanation of these results is that
the dephosphorylated NFAT1 is accessible to cellular kinases whether it
is located in the nucleus or in the cytoplasm. Chelation of
extracellular calcium with EGTA reversed the ionomycin-induced
dephosphorylation of NFAT1 with kinetics very similar to those seen for
reversal by CsA (Fig. 3A, compare lanes 4-6 with lanes 8-10), indicating that the
rephosphorylation was unlikely to be mediated by
Ca-dependent kinases, whose activity declines rapidly
after Ca
is withdrawn(49) .
Figure 4:
NFAT1
coprecipitates with calcineurin on CaM-Sepharose beads. A,
Ar-5 T cells (10 10
) were left
unstimulated(-) or were stimulated with ionomycin (+) in the
absence or presence of 1 µM CsA or FK506 and lysed in 1%
Triton X-100 buffer without added calcium (lanes 7 and 8) or supplemented with 1 mM CaCl
(lanes 1-6) or with 5 mM EGTA (lanes
9 and 10). Lysates were incubated with CaM-Sepharose
beads, and the bound proteins were analyzed by 7.5% SDS-PAGE followed
by Western blotting with a mixture of anti-NFAT1 and anti-calcineurin
antibodies. B, SDS lysates of unstimulated (UN) or
ionomycin-stimulated (IONO) Ar-5 T cells were analyzed by 7.5%
SDS-PAGE followed by Western blotting with anti-NFAT1 antibody (
NFAT; lanes 1 and 2). The blot was
stripped and incubated with
I-calmodulin (
I-CaM); the bands were visualized by
autoradiography (lanes 3 and 4). The blot was once
again stripped and reprobed with anti-calcineurin antibody (
Cn). C, Triton X-100 lysates of unstimulated Ar-5 T cells
without added calcium in the buffer were incubated with CaM-Sepharose
beads for three successive rounds of 1 h each at 4 °C. The lysate
was then supplemented with 100 µM or 1 mM CaCl
and subjected to one further incubation with
CaM-Sepharose beads before washing, boiling, and analysis as described
for A. Note that given the presence of EDTA, sodium
pyrophosphate, and NaF in the buffers, the free calcium concentration
in the buffers in lanes 5 and 6 is much lower than
the total concentration indicated and is plausibly in the physiological
range.
The binding of NFAT1 to CaM-Sepharose beads did not appear to reflect a direct interaction between NFAT1 and CaM. When a Western blot of whole cell lysates was used in an ``overlay'' experiment (39) with iodinated or biotinylated CaM, there was no detectable binding of CaM to NFAT1, even at high calcium concentrations (Fig. 4B, compare lanes 1 and 2 with lanes 3 and 4). In contrast, CaM bound strongly to calcineurin and to other unidentified CaM-binding proteins under the same conditions (compare lanes 3 and 4 with lanes 5 and 6). These results suggest that NFAT1 binds indirectly to CaM-Sepharose beads, through an ability to interact with calcineurin or other CaM-binding proteins. The specific involvement of calcineurin is suggested by the experiments of Fig. 5(B and C) below.
To investigate the calcium requirement
for binding of the putative NFAT1-calcineurin complex, we depleted
CaM-binding proteins from cell extracts by incubating them with
CaM-Sepharose beads in buffer without added Ca (Fig. 4C, lanes 1 and 2). This
process removed >95% of the calcineurin (and presumably other
CaM-binding proteins) capable of binding under these conditions
(compare lanes 1 and 2 with lanes 3 and 4). After an additional cycle of depletion (not shown),
Ca
was added to replicate samples of the depleted
lysates to achieve total Ca
concentrations of 100
µM and 1 mM (lanes 5 and 6,
respectively; again, the free Ca
concentrations in
these buffers are likely to be much lower and are plausibly in the
physiological range). These experiments showed a striking
Ca
-dependent increase in the level of NFAT1 bound to
the beads; concomitantly, an increase in the level of bound calcineurin
was observed (lanes 5 and 6). Addition of exogenous
calcineurin to the extracts did not increase the level of NFAT1 binding
to the CaM-Sepharose beads (data not shown). These results suggest that
the fraction of calcineurin that coprecipitates with NFAT1 under these
conditions has properties distinct from the bulk of the calcineurin in
that its binding to CaM-Sepharose requires higher concentrations of
calcium or trace concentrations of other ions present in our
Ca
stocks.
To address the possibility that NFAT1-(1-415) bound to CaM-beads in association with CaM-binding proteins other than calcineurin, we used a fusion protein of GST with the N-terminal 415 amino acids of NFAT (GST-NFAT1-(1-415)). GST-NFAT1-(1-415) did not bind to CaM-Sepharose directly, but was capable of binding in the presence of purified calcineurin and calcium (Fig. 5B, compare lane 1 with lanes 2 and 3). Bound GST-NFAT1-(1-415), which is a mixture of several proteolytic fragments formed by degradation within the bacteria (data not shown), was detected with an antibody to a peptide near the N terminus of NFAT1 (lane 1). Furthermore, consistent with the results of Fig. 4A, the association of purified calcineurin with GST-NFAT1-(1-415) was not disrupted by addition of CsA and purified cyclophilin A (Fig. 5C, lanes 2-4) or FK506 and purified FKBP12 (lanes 5-7). These experiments recapitulate the conditions of the earlier assay shown in Fig. 4A and Fig. 5A and support our interpretation that endogenous NFAT1 in cell lysates binds to CaM-Sepharose beads as a complex with calcineurin.
Although it has been well documented that CsA prevents the
calcineurin-mediated activation of
NFAT(15, 18, 19, 20) , thus far,
there has been no reason to expect that NFAT activation would be
continuously dependent on the level of calcineurin activity. We show
here that the activation of NFAT1 requires a sustained increase in the
level of cytoplasmic [Ca]
and
calcineurin activity in vivo. Inhibition of calcineurin by
addition of CsA or chelation of extracellular calcium by EGTA, even in
the constant presence of ionomycin, reverses NFAT1 activation by
permitting its rephosphorylation, its reappearance in the cytoplasm,
and the return of its DNA binding activity to a low level. It is likely
that all NFAT family proteins that are expressed at detectable levels
are similarly responsive to the level of calcineurin activity since CsA
completely inhibits the induction of nuclear NFAT-binding complexes in
a variety of immune system
cells(1, 15, 17, 18, 19, 20, 21, 22, 23) .
Our results are consistent with the ability of CsA to block ongoing
interleukin-2 transcription(13, 33, 45) .
They suggest a central function for NFAT family proteins in cytokine
gene transcription and explain the remarkable potency of CsA and FK506
as immunosuppressive drugs.
We have also shown that calcineurin
interacts directly with NFAT1 in vitro. A fraction of total
cellular calcineurin is coprecipitated with NFAT1 on
calmodulin-Sepharose beads under conditions distinct from those
required for binding of the bulk of calcineurin to the same beads. The
results are consistent with the hypothesis that a fraction of the
calcineurin is tethered to NFAT1 in a pre-existing complex; formation
of the complex may be stabilized at the higher cytoplasmic free calcium
concentrations that prevail in activated cells. The likelihood of an
NFAT1-calcineurin interaction provides an attractive explanation for
the sensitivity of NFAT1 to the level of calcineurin activity.
Moreover, it suggests that calcineurin dephosphorylates at least some
of the relevant phosphorylated residues of NFAT1, implying that NFAT1
is a physiological substrate for calcineurin and hence an immediate
secondary target of CsA and FK506 (1, 2, 50) . The N-terminal 415 amino acids
of NFAT1, which suffice for calcineurin binding, include the 300
amino acids of the NFAT homology region
; it remains to be
determined whether the conserved sequence motifs identified in this
region (8, 9, 10)
play a role in
calcineurin binding. The CaM-bound state of calcineurin is not required
for the interaction between calcineurin and NFAT1, as purified
calcineurin can be directly coprecipitated with GST-NFAT1-(1-415)
on glutathione-Sepharose beads. (
)In addition, the
NFAT1-calcineurin interaction observed in cell lysates is not affected
by pretreatment of cells with maximally immunosuppressive
concentrations of CsA and FK506. One explanation is that NFAT1 binds to
a region of calcineurin distinct from the B chain-binding
helix
that interacts with drug-immunophilin
complexes(51, 52) , thus rendering it feasible to
screen for drugs that block the NFAT1-calcineurin interaction without
interfering with calcineurin phosphatase activity.
Our results suggest that the phosphorylation state (and concomitantly the activation state) of NFAT1 is regulated by a dynamic equilibrium between calcineurin and basally active kinases. Tryptic phosphopeptide mapping indicates that NFAT1 is selectively dephosphorylated via calcineurin-dependent mechanisms, as at least two of several phosphopeptides of NFAT1 disappear when cells are stimulated with ionomycin. Moreover, these same two peptides reappear upon addition of CsA to ionomycin-stimulated cells, suggesting that kinases rapidly rephosphorylate NFAT1 following inhibition of calcineurin activity by CsA-cyclophilin. The phosphorylation state of several other phosphopeptides remains unchanged. It is possible that at least two classes of kinases act on NFAT1, one or more basally active kinases that phosphorylate the stimulation-insensitive sites and one or more kinases that phosphorylate only those sites that are dephosphorylated following T cell stimulation.
Our experiments do not address the intracellular location of the NFAT1-modifying enzymes in resting and activated cells. NFAT1 remains dephosphorylated and in the nucleus for long periods under conditions in which calcineurin activity is maintained, and dephosphorylated NFAT1 retains the ability to bind calcineurin directly; thus, one possibility is that a fraction of calcineurin translocates to the nucleus together with NFAT1. Likewise, dephosphorylated NFAT1 is accessible to kinases whether it is in the cytoplasm or the nucleus, suggesting either that the kinases are distributed throughout the cell or that they translocate with NFAT1 into the nucleus. However, it is equally possible that calcineurin and other relevant kinases and phosphatases function exclusively in the cytoplasm; if both dephospho-NFAT1 and phospho-NFAT1 constantly shuttled between the nucleus and the cytoplasm at rates determined by their phosphorylation state, both forms would be fully accessible to cytoplasmic enzymes.
The interaction of calcineurin with NFAT1 is a novel illustration of a more general principle, that the specificity of signaling pathways is achieved in many cases by targeting signaling proteins to their downstream effectors or substrates(34, 35, 36, 37, 38, 39, 40) . This targeting may be accomplished by stabilizing the normally transient interactions of enzymes with their substrates through protein-protein interactions, by using specialized binding domains such as SH2 and SH3 domains(36, 37) to recruit effector proteins, or by directing nonspecific enzymes to specific intracellular locations by means of targeting proteins or subunits(38, 39) . Several of these strategies may be utilized by the NFAT1-calcineurin complex; in particular, the NFAT1-calcineurin interaction is not dependent on the phosphorylation state of the substrate and thus resembles the stable interaction of Jun N-terminal kinase with both phosphorylated and dephosphorylated c-Jun proteins (34) . However, additional proteins may be involved in targeting, as shown for calcineurin itself and for other serine/threonine phosphatases(38, 39) . The catalytic subunit of protein phosphatase 1, which shows relatively low substrate specificity in vitro, is directed toward its substrates (the enzymes involved in glycogen metabolism) by a glycogen-binding targeting subunit(32, 38) . Likewise, the protein kinase A anchor protein AKAP-79 is a targeting protein that binds both to calcineurin and to protein kinase A(39) , thus bringing into proximity two enzymes that have mutually opposing actions on a variety of cellular processes(38) . By analogy, the NFAT1-calcineurin complex may include an additional targeting protein and also the kinases that deactivate NFAT1. ``Scaffolding'' proteins may also play a role. In yeast, multiprotein complexes comprising independent signaling modules, which contain enzymes associated with specific signaling pathways, have been described(40) , and similar large signaling complexes may regulate signal transduction in mammalian cells. Analysis of the NFAT1-calcineurin interaction represents the first step in delineating the regulatory components of this unique signaling pathway, in which calcineurin activation is directly coupled to inducible gene expression.
Note Added in Proof-An NFATI-calcineurin interaction has also been reported by Wesselborg et al.(54) .