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(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
The immunosuppressant drugs cyclosporin A (CsA) ( 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 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 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
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
A polyclonal antibody was developed against a peptide from
the COOH-terminal domain of NFAT Western blot analysis was carried out in order to
determine the expression of NFAT
Figure 1:
Expression of NFAT
The high level of NFAT Activation of T cells by
treatment with PMA and calcium ionophore stimulates NFAT DNA binding
activity(2) . The NFAT
Figure 2:
Ionomycin treatment of HT-2 cells results
in NFAT
To determine whether the changes in
NFAT A role for CaN in the
ionomycin-stimulated dephosphorylation of NFAT Previous studies demonstrated that
activation of T cells is accompanied by an increase in nuclear NFAT DNA
binding activity. Using the NFAT
Figure 4:
NFAT
The time
course for NFAT 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 Loss of NFAT
Figure 5:
[
During short labeling
times, the major NFAT 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 The NFAT Ionomycin treatment of the HT-2 cells
resulted in a significant shift in the molecular weight of
NFAT The results of the
localization experiments suggest that the phosphorylation state of
NFAT Following
ionomycin removal, NFAT The immunocomplex
assay demonstrated directly that NFAT 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
Volume 270,
Number 38,
Issue of September 22, pp. 22602-22607, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dephosphorylation and Nuclear
Localization in Activated HT-2 Cells Using a Specific NFAT
Polyclonal Antibody (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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.
)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) .
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) . 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., 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.
Materials
FK506 and rapamycin were prepared at
the Upjohn Company from fermentation broths of Streptomyces
tsukubaenis (Upjohn Culture Collection 11052) and Streptomyces
hygroscopicus (Upjohn Collection 5931), respectively. CaN and
calmodulin were obtained from Sigma, and PP2a was from UBI.
Orthophosphate was obtained from DuPont NEN.
Protein A-Sepharose CL-4B beads were from Pharmacia Biotech Inc.
[S]Methionine and ECL reagents were obtained
from Amersham Corp. Tyrosine phosphatase PTP1 was a gift from Dr. John
Bleasdale (The Upjohn Company). CaN autoinhibitory peptide (22) was synthesized at The Upjohn Company.
Cell Culture
The human T lymphoblastoid cell lines
J5D9 and HSB and the murine T lymphoma cell line EL-4 were grown in
RPMI 1640 medium containing 10% fetal calf serum, 2 mM
glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin.
The murine helper T cell line HT-2 was grown in the medium just
described with the addition of 12% rat T-STIM with concanavalin A as a
source of IL-2 (Collaborative Biomedical Products), 50 µM
-mercaptoethanol, and 10 mM HEPES, pH 7.4. Human
peripheral blood lymphocytes were prepared from leukophoresis packs.
Cells were centrifuged over Ficoll, and cells at the interface were
collected, diluted with RPMI, and pelleted. The cell pellet was
suspended in RPMI containing 10% fetal calf serum and incubated for 1 h
at 37 °C. Nonadherent cells were collected, pelleted, and stored at
-70 °C.Antibody Production
The amino acids CGG were added
to the amino terminus of the sequence LSPGAYPTVIQQQTAPSQR corresponding
to peptide 25 of NFAT(8) . The cysteine residue was
coupled to maleimide-activated KLH using the Imject Immunogen
Conjugation Kit (Pierce), and rabbits were injected. Sera was tested
against purified peptide, and bleeds determined to have the highest
titer were affinity-purified by using the Pierce Ag/Ab immobilization
kit with Sulfolink coupling gel. The antibody preparation used in the
experiments shown here recognized 1 ng of purified peptide at a 1:1000
dilution.Tissue Homogenates
Homogenates were prepared from
organs dissected from BALB/C mice as described in (20) using a
Polytron homogenizer. The tissues were homogenized at 0.3 g wet
weight/ml in 10 mM Tris, pH 8.0, containing 50 mM NaCl, 1 mM EDTA, 100 µg/ml aprotinin and soybean
trypsin inhibitor, 250 µM leupeptin, 10 mM
iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride. SDS was
added to bring the final concentration to 2%. The sample was heated at
100 °C for 10 min and then centrifuged for 10 min in a microfuge.
The supernatant was removed, and protein was determined by the
microassay method of Bradford (21) using reagents from Bio-Rad. Cellular Fractionation
Treatment of HT-2 cells (1
10
cells/condition) was carried out as described in
the figure legends. The cells then were washed in ice-cold PBS and
resuspended (2.5 10 cells/ml) in buffer (10 mM Tris-Cl, pH 7.5, containing 10 mM NaCl, 3 mM
MgCl
, 1 mM phenylmethylsulfonyl fluoride, 0.5
mM dithiothreitol, 0.1 mM EGTA, 2 µM leupeptin, 1 µg/ml aprotinin, and 0.05% Nonidet P-40). Cells
were centrifuged at 650 g to pellet the nuclei, and
the supernatant was removed. The nuclear pellet was washed in the above
buffer minus detergent, and the final pellet was resuspended in Laemmli
buffer. The supernatant was recentrifuged to remove any residual
nuclei. Equal cell equivalents of soluble and nuclear fractions were
electrophoresed on 6% SDS-polyacrylamide gels, and Western blotting was
carried out as described below.Western Blots
Cells were treated (2.5
10
cells/condition) as indicated in the figure legends, and
control cells received MeSO as a vehicle. Following
treatment, the cells were washed with ice-cold PBS and resuspended in
40 mM Tris, pH 7.5, containing 10 mM EDTA, and 60
mM sodium pyrophosphate. SDS was added to bring the final
concentration to 5%. The sample was boiled for 15 min and
electrophoresed on a 6% SDS-polyacrylamide gel. Proteins were
transferred to nitrocellulose, and nonspecific sites were blocked with
5% nonfat dry milk in PBS. The blot was developed with a 1:500 dilution
of affinity-purified NFAT antibody in 20 mM Tris,
pH 7.5, containing 500 mM NaCl and 3% bovine serum albumin for
1 h at room temperature followed by incubation with a 1:5000 dilution
of donkey anti-rabbit IgG linked to horseradish peroxidase for 1 h
(Amersham Corp.). Immunoreactive proteins were detected with the ECL
system. For the phosphotyrosine blot (Fig. 3B), lysate
from untreated HT-2 cells or epidermal growth factor-treated A431 cells
(300 ng/ml epidermal growth factor for 15 min) were probed with 1
µg/ml 4G10 antibody (UBI), and the blot was developed by ECL.
, 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.
HT-2 cells
at a density of 1 P Immunoprecipitation
10 cells/ml were grown overnight
in growth medium as described above except that the medium contained 2%
of the usual phosphate concentration and 10% dialyzed fetal calf serum.
The cells were resuspended in fresh medium containing 100 µCi/ml
[P]orthophosphate. Cells were aliquoted at 1.5
10 cells/condition, and treatments were carried out
as described in the figure legends. The cells were allowed to
incorporate radiolabel for a total time of 4 h. After 2.8 h of
incubation, FK506 (500 nM) was added for 1 h, followed by
ionomycin (2 µM) for an additional 10 min. The cells were
washed in ice-cold PBS and lysed in radioimmune precipitation buffer
(10 mM Tris, pH 7.5, containing 1% Triton X-100, 1%
deoxycholate, 0.1% SDS, and 150 mM NaCl) containing 60 mM sodium pyrophosphate and 10 mM EDTA. The sample was
precleared with Protein A-Sepharose CL-4B beads for 30 min at 4 °C.
SDS was added (0.3% final concentration), and 10 µl of
affinity-purified antibody was added to the sample for 2 h at 4 °C
followed by Protein A for 1 h. The Protein A beads were washed three
times in radioimmune precipitation buffer containing 500 mM NaCl and 2 mg/ml bovine serum albumin, followed by three washes in
radioimmune precipitation buffer containing 0.3% SDS, followed by a
final wash in radioimmune precipitation buffer. The sample was
resuspended in Laemmli buffer and electrophoresed on a 6%
SDS-polyacrylamide gel followed by autoradiography. The autorad was
quantitated on a Molecular Dynamics PhosphorImager using ImageQuant
Software.Immunocomplex Assays
Unlabeled cells (1.5
10 cells/condition) were untreated or stimulated for 10 min
with 2 µM ionomycin, immunoprecipitated as described
above, and washed five times in assay buffer (40 mM Tris, pH
7.5, containing 100 mM NaCl, 0.5 mM CaCl,
0.5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 1
mM MgCl, and 0.1 mM MnCl).
The immunoprecipitate from untreated cells was incubated in assay
buffer containing the enzymes and inhibitors as indicated in the Fig. 3A legend in a final volume of 100 µl for 30
min at 30 °C. 50 µl of Laemmli buffer was added, the sample was
boiled, and the Sepharose beads were pelleted. The supernatant was
electrophoresed on a 6% SDS-polyacrylamide gel, and Western blotting
was carried out as above.Phosphatase Assays
The phosphatase activity of CaN
and PP2A was measured in a total volume of 100 µl containing 40
mM Tris, pH 8.6, 100 mM NaCl, 0.5 mM CaCl, 0.5 mM dithiothreitol, 0.1 mg/ml bovine
serum albumin, 10 mM MgCl, 600 µM 4-methylumbelliferryl phosphate and either 25 nM CaN or
0.1 units of PP2A. To measure CaN activity, assays also included 100
nM calmodulin and the presence or absence of 100
µM inhibitory peptide(22) . The mixture was
incubated for 1 h at 37 °C followed by the addition of 50 µl of
stop buffer (300 mM glycine, pH 11.2, containing 15 mM EDTA), and the fluorescence was read at 365/405 nm.[
Cells were resuspended at 3 S]Methionine
Pulse-Chase
10 cells/ml for 30 min in growth medium containing 2% of the usual
methionine plus 10% dialyzed fetal calf serum, in the presence or
absence of 2 µM ionomycin.
[S]Methionine was added at 175 µCi/ml for 5
min, and 1.5
10 cells were removed for the zero
time point. The remainder of the cells were washed in PBS and
resuspended in normal growth medium in the presence or absence of 2
µM ionomycin. At the indicated times, aliquots (1.5
10 cells) were removed, lysed, and
immunoprecipitated with the NFAT antibody as described
above. The autorad was quantitated as described above.[
Cells were resuspended at 6 S]Methionine
Degradation
10 cells/ml in low methionine-containing medium plus
[S]methionine (50 µCi/ml) and incubated for
18 h. The cells were washed in PBS and resuspended at 3
10 cells/ml in normal growth medium containing
MeSO (control), 2 µM ionomycin, or 100 nM FK506 plus 2 µM ionomycin. Cells were pretreated with
FK506 5 min prior to the addition of ionomycin. Cells (1.5
10) were removed immediately (zero time point) or at the
indicated times, and the lysates were immunoprecipitated as described
above. Autorads were quantitated as described above and analyzed by
linear regression to determine the NFAT half-life.
. 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. 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.
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.
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). 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).
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.
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. 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 NFATin 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. 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).
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).
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. 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. 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.
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.
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.
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. 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., 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. 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. 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. 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.-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.
)
We acknowledge Dr. Anjana Rao for the gift of Ar-5
extracts, Kay Petruska for secretarial assistance, and Dr. Clark Smith
and Carol Bannow for peptide synthesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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