(Received for publication, June 26, 1995)
From the
Nuclear factor of activated T cells (NFAT) regulates
transcription of a number of cytokine genes, and NFAT DNA binding
activity is stimulated following T cell activation. Several lines of
evidence have suggested that NFAT is a substrate for calcineurin, a
serine/threonine phosphatase. Using a polyclonal antibody to murine
NFAT, Western blot analysis of various mouse tissues
demonstrated that the 110-130-kDa NFAT
protein was
highly expressed in thymus and spleen. Treatment of immunoprecipitated
NFAT
from untreated HT-2 cells with calcineurin resulted in
the dephosphorylation of NFAT
, demonstrating that
NFAT
is an in vitro substrate for calcineurin.
NFAT
immunoprecipitated from
P-labeled HT-2
cells migrated as an approximately 120-kDa protein that was localized
to the cytosol of the cells. Treatment of the cells with ionomycin
resulted in a decrease in the molecular weight of NFAT
and
a loss of
P, consistent with NFAT
dephosphorylation. The dephosphorylation of NFAT
was
accompanied by localization of the protein to the nuclear fraction.
Both of these events were blocked by preincubation of the cells with
FK506, a calcineurin inhibitor, consistent with the hypothesis that
NFAT
is a calcineurin substrate in cells.
The immunosuppressant drugs cyclosporin A (CsA) ()and
FK506 inhibit the early steps of antigen-stimulated T cell activation.
These drugs prevent activation of a variety of cytokine genes,
including IL-2, granulocyte-macrophage colony-stimulating factor, IL-4,
and tumor necrosis factor
(for review, see (1) and (2) ). IL-2 plays a key role in controlling T cell
proliferation, and consequently, numerous studies have focussed on
understanding IL-2 gene regulation. The results of these studies
demonstrated that transcriptional activation requires nuclear factor of
activated T cells (NFAT), a DNA binding protein, which binds to
specific sites on the regulatory regions of the cytokine
genes(3, 4, 5, 6) .
Molecular
cloning and biochemical studies have provided insights into the actions
of NFAT. The approximately 120-kDa NFAT is a member of a gene family
whose members appear to contain a Rel homology region, which is
important for DNA binding activity. Two members were identified:
NFAT was cloned from a human T cell library (7) ,
while NFAT
was purified and cloned from murine T
cells(8) . In addition, several alternatively spliced forms of
NFAT
were identified(2) . NFAT
and
NFAT
are approximately 73% identical in the Rel homology
region, although they share little similarity outside that domain. In
unactivated T cells, NFAT DNA binding activity is localized primarily
in the cytosol, and following T cell activation, activity is detected
in the nucleus. Nuclear NFAT forms a complex with Fos and Jun, and
mutations in NFAT that inhibit binding to these proteins eliminate
NFAT-mediated gene transcription. Furthermore, treatment of cells with
either CsA or FK506 inhibits the appearance of NFAT binding activity in
the nucleus(3, 9, 10, 11) .
An understanding of the link between the drugs FK506 and CsA and NFAT has recently started to emerge with the identification of calcineurin (CaN) as a cellular target of the drugs. FK506 and CsA bind to their respective immunophilins, FKBP-12 and cyclophilin A, and the drug-immunophilin complexes bind to and inhibit the activity of CaN, a calmodulin-dependent phosphatase(12, 13) . Overexpression of a constitutively active form of CaN in T cells renders the cells more resistant to the effects of the drugs, and the ability of a number of FK506 and CsA analogues to inhibit the phosphatase activity of CaN correlates with their ability to inhibit T cell activation(14) .
Evidence suggests that CaN is not just involved in mediating the actions of FK506 and CsA but that the enzyme plays a role in the physiological pathway of T cell activation. O'Keefe, et al.(15) demonstrated that in cells transfected with CaN, PMA- and ionophore-stimulated IL-2-driven gene expression was stimulated approximately 2-fold. Furthermore, in cells overexpressing a constitutively active form of CaN, IL-2-driven gene expression was stimulated approximately 150-fold in the presence of PMA alone, indicating that active CaN can replace the usual requirement for ionophore. Similar results were obtained by Clipstone and Crabtree (16) , who demonstrated reporter gene activity in CaN-transfected cells at suboptimal concentrations of ionomycin that elicited little or no activity in control cells. Taken together, these results demonstrate that CaN overexpression augments T cell activation resulting from both calcium and PKC stimulation and suggest that CaN plays a key role in this process.
The studies with CsA and FK506 led
to the suggestion that NFAT is a CaN substrate, either direct or
indirect, in T cells. First, indirect evidence indicates that
NFAT is a phosphoprotein. Fractionation of Jurkat cell
cytosol demonstrated that NFAT DNA binding activity is detected with
proteins in a molecular mass range of 94-116 kDa, suggesting
heterogeneity in the size of the NFAT protein (17) . To
determine whether NFAT phosphorylation could explain the heterogeneity,
McCaffrey, et al.(18) treated cell extracts enriched
for NFAT with alkaline phosphatase. This enzymatic treatment resulted
in a shift in NFAT binding activity from the 127-143-kDa
molecular weight fraction to a 101-113-kDa fraction, which is
consistent with the hypothesis that NFAT is a phosphoprotein. Second,
treatment of purified NFAT with purified bovine brain CaN also resulted
in a shift in the molecular weight of NFAT, suggesting that NFAT is an in vitro substrate for CaN(19) . Whether NFAT is a
direct CaN substrate in intact cells has not yet been determined,
however.
In the majority of reported NFAT studies, NFAT DNA binding
activity has been the end point usually measured, since relatively
little is known about the NFAT protein. However, the recent cloning and
expression of NFAT genes has yielded valuable information about the
proteins, and direct protein analyses should now be possible. Towards
that end, antibodies to NFAT will be important reagents to develop for
probing protein structure and function. In the studies reported here,
we developed a peptide antibody to NFAT, and we used it to
characterize NFAT
in cells. We demonstrate directly in
P labeling experiments that NFAT
is a
phosphoprotein and that T cell activation results in NFAT
dephosphorylation that can be blocked by pretreatment with FK506.
Furthermore, we show that dephosphorylation is accompanied by
translocation of NFAT
protein from the cytosol to the
nucleus of cells.
Figure 3:
Immunocomplex phosphatase assay and
phosphotyrosine blot. A, immunocomplex phosphatase assay.
Immunoprecipitates from untreated cells (lane1) and
ionomycin-treated cells (lane2) are shown.
Immunoprecipitates were prepared from untreated cells as described
under ``Experimental Procedures'' and incubated with the
following: 50 nM CaN plus 200 nM calmodulin (lane3), 50 nM CaN, 200 nM calmodulin and
500 µM autoinhibitory peptide (lane 4), 0.1 units PP2a (lane5), PP2a plus 500 nM okadaic acid (lane6), PTP1 (lane7), and 50
nM CaN and 200 nM calmodulin (lane8), developed with second antibody alone. The thick
arrow points to the phosphorylated form of NFAT, and
the thin arrow points to the dephosphorylated form. B, phosphotyrosine blot. Lane1, HT-2 cell
lysate; lane2, A431 cell lysate. The
170-kDa
epidermal growth factor receptor is shown in lane2 as a positive control for the phosphotyrosine
antibody.
A polyclonal antibody was developed against a peptide from
the COOH-terminal domain of NFAT. This peptide is outside
the Rel homology domain and is from a unique region of NFAT
that is not present in NFAT
(7) . Therefore,
this antibody is a specific reagent for characterizing the NFAT
protein.
Western blot analysis was carried out in order to
determine the expression of NFAT in various mouse tissues (Fig. 1A). The highest level of NFAT
expression was observed in spleen and thymus tissue. In thymus,
the antibody strongly hybridized with proteins in the 110-130-kDa
molecular mass range, suggesting protein heterogeneity, while in spleen
tissue, reactivity was predominantly against a 110-kDa protein. In both
these tissues, reactivity with smaller proteins of 72, 66, and 59 kDa
was observed. It is unknown whether these proteins are proteolytic
fragments of the larger NFAT
protein. However, in
competition experiments in which the NFAT
peptide was
included in the antibody incubation, no reactivity was observed,
indicating the specificity of the interactions (lanes8 and 9). In kidney and liver, there was little or no
antibody reactivity against proteins in the 120-kDa range.
Cross-reactivity against proteins of 66 and 72 kDa was shared among
kidney, spleen, and thymus, although interestingly, in spleen and
thymus, reactivity against the 66-kDa protein was predominant, while in
kidney, antibody reactivity was greater against the 72-kDa protein,
compared with the 66-kDa protein. Expression of NFAT
was
very low in brain, while in heart no NFAT
was detected.
Figure 1:
Expression of NFAT in
mouse tissues and T cells. Aliquots of mouse tissue homogenate or cell
lysates were prepared, electrophoresed, transferred to nitrocellulose,
and probed with the NFAT
antibody as described under
``Experimental Procedures.'' The position of the molecular
weight markers is as indicated. A, 200 µg of mouse tissue
homogenate from brain (lane1), heart (lane2), kidney (lane3), liver (lane4), spleen (lane5), thymus (lane6), and 2.5
10
HT-2 cell equivalents
as a control (lane7). Lanes8 and 9 contain spleen and thymus, respectively, and the blot was
developed using antibody containing a 100-fold excess of the antigen
peptide. B, 1
10
cell equivalents from
J5D9 (lane1), HSB (lane2), EL-4 (lane3), peripheral blood lymphocytes (lane4), 2.5
10
cell equivalents from HT-2
cells (lane5), 1
10
cell
equivalents from EL-4 cells (lane6), and 2.5
10
cell equivalents from HT-2 cells (lanes7 and 8). Lanes6 and 7 were
developed with antibody in the presence of antigen peptide, and lane8 was developed with second antibody
alone.
The high level of NFAT expression in spleen and thymus
is consistent with previous reports demonstrating NFAT DNA binding
activity in T cells. Various T cell lines were profiled to investigate
whether NFAT
levels differ among T cells (Fig. 1B). Peripheral blood lymphocytes had the lowest
level of NFAT
expression (lane4). These
results could not simply be explained by a lack of reactivity of the
murine peptide-derived antibody against human protein, since human T
cell Jurkat cells showed significant reactivity with the antibody (lane1). HSB cells, a human T cell line, also
expressed the approximately 140-kDa NFAT
(lane2). In EL-4 cells, a mouse thymoma cell line, NFAT
was expressed, but the protein was more heterogeneous, since
proteins from 120 to 140 kDa reacted with the antibody (lane3). HT-2 cells, an IL-2-dependent mouse T cell line, had
the highest level of NFAT
expression compared with the
other cell lines (lane5). In the experiment shown in Fig. 1B, lysate from 2.5
10
HT-2
cells resulted in a comparable or greater signal, compared with the
other cell lines, in which 4-fold more cell equivalents (1
10
) were used. In all of the cell lines, cross-reactivity
with a 66-kDa protein was seen, suggesting, as in the tissue samples,
that other NFAT
protein forms may be expressed. Competition
experiments using NFAT
peptide and EL-4 or HT-2 cell
extracts (lanes6 and 7) demonstrated that
all the reactivity could be competed by excess peptide. No NFAT protein
was detected in MOLT-4 cells, a human T cell leukemia line or in NIH
3T3 cell fibroblasts (data not shown).
Activation of T cells by
treatment with PMA and calcium ionophore stimulates NFAT DNA binding
activity(2) . The NFAT antibody was used to
investigate directly the effect of T cell activation on NFAT protein
levels (Fig. 2A). For these and subsequent experiments,
HT-2 cells were used, due to their high level of expression of
NFAT
. NFAT
migrated as an approximately 120-kDa
protein in untreated cells. Treatment of the cells with either
ionomycin alone (lane2) or the combination of PMA
plus ionomycin (data not shown), resulted in a decrease in the
molecular weight of NFAT
, suggestive of a proteolytic
and/or dephosphorylation event. Identical shifts in the NFAT
protein were observed when the cell treatments and lysate
preparation were carried out in the presence of a panel of protease
inhibitors, including leupeptin, phenylmethylsulfonyl fluoride, and
1-chloro-3-tosylamido-7-amino-2-heptanone (data not shown), suggesting
that the mobility shift was not the result of proteolysis. The
predominant NFAT
protein band in the ionomycin-treated
cells was approximately 110 kDa, although the protein was heterogenous
in size. Pretreatment of the cells with 500 nM FK506 blocked
the ionomycin-induced shift in NFAT
(lane3), suggesting a role for CaN. As a control, cells were
pretreated with rapamycin prior to ionomycin treatment. Rapamycin binds
to FKBP-12, but unlike FK506, it does not inhibit CaN, nor does it
inhibit NFAT DNA binding activity. Rapamycin pretreatment also did not
block the NFAT
mobility shift induced by ionomycin (lane4).
Figure 2:
Ionomycin treatment of HT-2 cells results
in NFAT dephosphorylation. A and B, HT-2
cells were treated and lysates were prepared for Western blotting (A), or NFAT
was immunoprecipitated from
P-labeled cells (B) as described under
``Experimental Procedures.'' Lane1,
control; lane2, 2 µM ionomycin for 10
min; lane3, 500 nM FK506 pretreatment (1 h)
followed by 2 µM ionomycin for 10 min; lane4, 1 µM rapamycin pretreatment (1 h)
followed by ionomycin for 10 min.
To determine whether the changes in
NFAT mobility were the result of ionomycin-induced
dephosphorylation,
P labeling experiments were carried out (Fig. 2B). Cells were labeled with
[
P]orthophosphate for 4 h followed by treatments
similar to those shown in Fig. 2A, and
immunoprecipitation was carried out with the NFAT
antibody.
A similar decrease in molecular weight was observed in the
P-labeled NFAT
following ionomycin treatment
as was observed in the Western blot experiments. Quantitation of the
P autoradiograms showed that ionomycin treatment resulted
in an approximately 65% loss in
P from NFAT
(data not shown). Interestingly, ionomycin treatment never
resulted in a complete loss of
P from NFAT
,
indicating that complete dephosphorylation did not occur. In addition,
FK506 inhibited the loss of
P, while rapamycin had no
effect. The results of these experiments demonstrate directly that the
shift in molecular weight following ionomycin treatment was the result
of NFAT
dephosphorylation and that FK506 pretreatment
blocked the dephosphorylation.
A role for CaN in the
ionomycin-stimulated dephosphorylation of NFAT was shown
directly in immune complex assays (Fig. 3). NFAT
was
immunoprecipitated from untreated HT-2 cells, and the
immunoprecipitates were washed and incubated with CaN. CaN
dephosphorylated NFAT
in vitro, as shown by a
decrease in the NFAT
molecular weight, which was comparable
to the molecular weight shift resulting from treatment of cells with
ionomycin (compare lanes2 and 3). CaN
contains an autoinhibitory domain in its COOH-terminal domain, and a
peptide from this region inhibits CaN dephosphorylation of a
cAMP-dependent protein kinase peptide in vitro ( (22) and data not shown). Addition of the autoinhibitory
peptide to the immune complex assay blocked the dephosphorylation of
NFAT
by CaN (lane4). In contrast,
treatment of the precipitated NFAT
with another
serine/threonine phosphatase, PP2A, resulted in little or no
dephosphorylation of NFAT
(lane5),
although the enzyme readily dephosphorylated 4-methylumbelliferryl
phosphate (data not shown). The tyrosine phosphatase PTP1 also did not
dephosphorylate NFAT
(lane7), suggesting
that NFAT
may not contain phosphorylated tyrosine residues.
Consistent with this hypothesis was the observation that there was no
reactivity on Western blots of antiphosphotyrosine antibodies with
NFAT
(Fig. 3B). Taken together, these
results indicate that NFAT
readily serves as an in
vitro substrate for CaN.
Previous studies demonstrated that
activation of T cells is accompanied by an increase in nuclear NFAT DNA
binding activity. Using the NFAT antibody, we showed
directly the presence of the NFAT
protein in the nucleus of
stimulated cells (Fig. 4A). In untreated cells, all of
the NFAT
protein was detected in the low speed supernatant (lane1). Following a 10-min ionomycin treatment,
however, the lower molecular weight, dephosphorylated form of
NFAT
was predominantly localized to the nuclear fraction (lane5). Pretreatment of cells with FK506 prior to
ionomycin stimulation blocked the dephosphorylation, as previously
observed, and also blocked the translocation of NFAT
to the
nucleus (compare lanes3 and 6).
Figure 4:
NFAT is localized to the
nucleus in ionomycin-treated cells. A, HT-2 cells were
treated, and soluble and nuclear fractions were prepared and
Western-blotted as described under ``Experimental
Procedures.'' Lanes1-3 are soluble
fractions, and lanes4-6 are nuclear fractions. Lanes1 and 4, control; lanes2 and 5, 2 µM ionomycin for 10 min; lanes3 and 6, 500 nM FK506
pretreatment (1 h) followed by ionomycin for 10 min. B,
extended ionomycin time course; C, ionomycin washout time
course. For the experiment shown in panelB, cells
were stimulated with 2 µM ionomycin, and the zero time
aliquot (1
10
cells) was removed immediately after
the addition of ionomycin. For the experiment shown in panel
C, cells were stimulated with 2 µM ionomycin for 10
min. The cells were washed in PBS and resuspended at 2
10
cells/ml in normal growth medium, and an aliquot (1
10
cells) was removed immediately for the zero time point. Lanes1 and 2 contain lysate prepared from
2.5
10
cells from untreated or ionomycin-treated
cells, respectively, as controls. Supernatant (upperpart) and nuclei (lowerpart) are shown
from untreated cells (lane3) and cells treated with
ionomycin for the indicated times: 0 time (lane4),
10 min (lane5), 30 min (lane6), 1
h (lane7), 2 h (lane8), 3 h (lane9), and 4 h (lane10).
The time
course for NFAT dephosphorylation and nuclear localization
was examined in more detail (Fig. 4B). Fractionation of
cells immediately following the addition of ionomycin indicated that
NFAT
dephosphorylation was rapid (lane4), as shown by the ladder of protein bands. After
ionomycin treatment for 10 min, the dephosphorylated NFAT
was present in both the low speed supernatant and the nuclear
fractions (lane5), suggesting that NFAT
is dephosphorylated in the cytosol, followed by localization of
the dephosphorylated form to the nucleus. With increasing time of
incubation with ionomycin, dephosphorylated NFAT
was
present almost exclusively in the nuclear fraction, although a small
level of dephosphorylated NFAT
was detected in the
supernatant throughout the 4-h time course. These results suggest that
NFAT
is rapidly dephosphorylated and localized to the
nucleus following ionomycin treatment and that the dephosphorylated
NFAT
remains in the nucleus in the continuing presence of
ionomycin.
A similar localization experiment was carried out with
cells pretreated with ionomycin, followed by removal of the ionomycin (Fig. 4C). At the start of the experiment, following a
10 min treatment with ionomycin, essentially all of the NFAT was localized to the nucleus, and the nuclear NFAT
was in the lower molecular weight, dephosphorylated form. After
10 min following washout of the ionomycin, NFAT
was still
in the nuclear fraction, although a low level of the higher molecular
weight, phosphorylated form of NFAT
was detected in the low
speed supernatant. However, at the 30-min time point and beyond,
NFAT
was localized in the low speed supernatant fraction,
with no detectable NFAT
in the nucleus. These results
suggest that following ionomycin washout, NFAT
is recycled
from the nucleus back into the cytosol. Furthermore, the NFAT
in the low speed supernatant was exclusively in the higher
molecular weight, phosphorylated form, suggesting that migration from
the nucleus was accompanied by rapid rephosphorylation of
NFAT
.
Loss of NFAT from the nuclear fraction
could result from NFAT
degradation. To investigate this
possibility, [
S]methionine labeling experiments
were carried out to measure the half-life of the NFAT
protein. Cells were labeled overnight with
[
S]methionine and then chased in medium
containing unlabeled methionine. The rate of loss of immunoprecipitated
NFAT
was determined for untreated, ionomycin-treated, and
FK506- and ionomycin-treated cells, and is shown as a semi-log plot (Fig. 5A). In untreated cells the t for
NFAT
was 16.9 ± 0.86 h (mean ± S.E., n = 3), which was similar to the value obtained from cells
treated with the combination of FK506 and ionomycin (19.2 ± 3.9
h). Treatment with ionomycin, however, caused a slight decrease in the
time required for loss of 50% of the prelabeled NFAT
, to
11.9 ± 2.7 h. These results indicate that the loss of NFAT
from the nucleus was not the result of degradation of the protein
and support the hypothesis that nuclear NFAT
reappears in
the cytosol following ionomycin removal.
Figure 5:
[S]methionine
labeling experiments. A, degradation. The data points shown
are the mean ± S.E. of three experiments. HT-2 cells were
labeled and treated as described under ``Experimental
Procedures.''
, control;
, ionomycin;
, FK506
plus ionomycin. The inset shows a representative autorad from
untreated cells. B, pulse-chase experiment. Lanes1-3, immunoprecipitates from untreated cells; lanes4 and 5, immunoprecipitates from
ionomycin-treated cells; lanes1 and 4, 0
time; lanes2 and 5, 10 min; lane3, 20 min.
During short labeling
times, the major NFAT was in the dephosphorylated form (Fig. 5B). However, this form was rapidly chased into
the higher molecular weight, phosphorylated form of NFAT
,
suggesting that phosphorylation of NFAT
occurs very rapidly
following synthesis.
Much of the available information concerning NFAT is the
result of studies of NFAT DNA binding
activity(1, 2, 3, 9, 11) .
More recently, several forms of NFAT have been cloned, and Northern
analysis demonstrated that NFAT mRNA levels vary among different
tissues and under stimulus conditions(7) . Investigations with
a specific NFAT antibody allowed expansion of these studies
to include direct analysis of the NFAT
protein. The peptide
sequence used to generate the antibody described here is derived from
the COOH-terminal region of NFAT
, which is not present in
NFAT
, and thus the antibody differentiates between the two
NFAT forms.
The NFAT protein was highly expressed in
thymus and spleen tissue, which is consistent with mRNA
analysis(7) . However, Northrup et al.(7) reported that NFAT
mRNA levels were
similar in brain, heart, thymus, and spleen, while our immunoblotting
results (Fig. 1) showed that no NFAT
protein was
detected in heart tissue, and only very low levels in brain. These
results suggest that the presence of NFAT
mRNA may not
predict the expression of the protein in all tissues or cells. In
addition, lower molecular weight proteins reacted with the NFAT
antibody, suggesting that other forms of NFAT
may
exist. Consistent with these results is the report that COOH-terminal
splicing variants of NFAT
have been cloned(2) .
Peptide mapping or purification of the lower molecular weight,
cross-reactive proteins is required to determine whether they are
related to NFAT
.
Ionomycin treatment of the HT-2 cells
resulted in a significant shift in the molecular weight of
NFAT, as shown by immunoblotting analysis. The
P labeling experiments provided a direct demonstration
that NFAT
is a phosphoprotein and that the shift in
molecular weight resulted from dephosphorylation of NFAT
.
The dephosphorylation-induced molecular weight mass was approximately
10 kDa and represented an approximately 65% loss in phosphate from the
protein. The extent of phosphorylation of NFAT
is not
known, and the results shown here indicate that dephosphorylation
results in a significant change in the mobility of the protein on
SDS-polyacrylamide gels. This result has practical applications, for it
allows detection of the phosphorylated and dephosphorylated forms of
NFAT
using Western blot analysis.
The results of the
localization experiments suggest that the phosphorylation state of
NFAT may be a determinant of its localization within the
cell. In untreated cells, NFAT
was in the phosphorylated
state, and was localized to the low speed supernatant. However, in
ionomycin-treated cells, dephosphorylated NFAT
was found in
the nuclear fraction. Kinetic analysis, shown in Fig. 4,
suggests that dephosphorylation occurs prior to nuclear localization,
since at the 10-min time point, dephosphorylated NFAT
was
present in both the cytosolic and nuclear fractions, while at later
time points the dephosphorylated form was exclusively localized to the
nucleus. As long as ionomycin was present, NFAT
remained
both dephosphorylated and in the nucleus. Previous studies demonstrated
that NFAT
DNA binding activity is localized to the cytosol
of untreated T cells, and to the nuclear fraction in stimulated
cells(3) . The results shown here demonstrate directly that the
shift in DNA binding activity seen by others reflects a change in the
cellular location of the NFAT
protein.
Following
ionomycin removal, NFAT reappeared in the cell supernatant
fraction, in the higher molecular weight, phosphorylated form. The
[
S]methionine labeling experiments demonstrated
that NFAT
is a relatively stable protein, with a half-life
of approximately 17 h in untreated cells and approximately 12 h in
ionomycin-treated cells. Thus, the loss of signal from the nucleus
following ionomycin removal cannot be explained by degradation of
NFAT
. These results suggest that NFAT
may
shuttle in and out of the nucleus, depending on the activation state of
the cell as well as the phosphorylation state of the protein. A
potential nuclear localization sequence has been identified within the
Rel homology region of NFAT
(23) . There are a
number of potential phosphorylation sites in NFAT
,
including one within the nuclear localization sequence. One possibility
is that activation-induced dephosphorylation of NFAT
results in an unmasking of the nuclear localization sequence,
leading to nuclear localization of the protein.
The immunocomplex
assay demonstrated directly that NFAT is a CaN substrate in vitro. Furthermore, the CaN-induced molecular weight shift
in NFAT
was similar to that resulting from ionomycin
treatment of cells. Treatment of intact cells with FK506, a CaN
inhibitor, blocks the ionomycin-induced dephosphorylation, which
further supports but does not prove the hypothesis that NFAT
is a CaN substrate in intact cells.
In general, no consensus
sequence for dephosphorylation of substrates by CaN has been
identified. However, Donella-Deana et al.(24) have
demonstrated in phosphopeptide studies that basic residues on the
NH-terminal side of the phosphoamino acid, especially at
the -3 position, are positive determinants for dephosphorylation
by CaN, while acidic residues on the COOH-terminal side are negative
determinants. Because of a lack of a strong consensus sequence, these
results and the results of others (25, 26) have led to
the suggestion that more complex protein structural determinants also
play a role in defining CaN substrate specificity. Taken together,
these results suggest that a number of sites within NFAT
may be sites for CaN dephosphorylation. Mapping these sites in vitro and comparing them with the sites of
dephosphorylation in intact cells is required to establish whether
NFAT
is a direct substrate for CaN in intact cells. The
results of such experiments will define in more detail the exact role
of CaN in T cell activation.