(Received for publication, April 4, 1995; and in revised form, June 12, 1995)
From the
Cyclosporin A (CsA) exerts its immunosuppressive effect by
inhibiting the activity of nuclear factor of activated T cells (NFAT),
thus preventing transcriptional induction of several cytokine genes.
This effect is thought to be largely mediated through inactivation of
the phosphatase calcineurin, which in turn inhibits translocation of an
NFAT component to the nucleus. Here we report that CsA treatment of
Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp
that is inhibited in DNA-binding and in its ability to form an NFAT
complex with Fos and Jun. Immunoblot analyses and metabolic labeling
with [P]orthophosphate show that CsA alters
NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing
its phosphorylation level without affecting subcellular distribution.
Dephosphorylation by in vitro treatment with calcineurin or
alkaline phosphatase restores NFATp DNA binding activity and its
ability to reconstitute an NFAT complex with Fos and Jun proteins.
These data point to a new mechanism for CsA-sensitive regulation of
NFATp in which dephosphorylation is critical for DNA binding.
The immunosuppressive drugs cyclosporin A (CsA) ()and
FK-506 repress an early event in T cell activation by inhibiting a
step(s) in intracellular calcium signaling, thus preventing the
transcription of several cytokine genes including interleukin (IL)-2,
IL-3, IL-4, granulocyte/macrophage colony-stimulating factor, tumor
necrosis factor-
, and other molecules that play a major role in
coordinating the immune response (for review, see Sigal and
Dumont(1992), Schreiber and Crabtree (1992), and Bierer et
al.(1993)). Although the mechanism(s) by which these drugs inhibit
transcription of a discrete set of cytokines is not yet well defined,
recent studies have implicated the
Ca
/calmodulin-dependent protein phosphatase,
calcineurin (CaN), as an intracellular component responsible for
regulating the drug-sensitive expression of at least the IL-2, tumor
necrosis factor-
, and granulocyte/macrophage colony-stimulating
factor genes (Clipstone and Crabtree, 1992; O'Keefe et
al., 1992; Goldfeld et al., 1994; Tsuboi et al.,
1994). Nuclear factor of activated T cells (NFAT) is involved in the
transcription of these cytokines and may be a major target of the
activity of CaN and the immunosuppressive drugs (Emmel et al.,
1989; Granelli-Piperno et al., 1990; Randak et al.,
1990; Chuvpilo et al., 1993; Cockerill et al., 1993;
Goldfeld et al., 1993; Masuda et al., 1993; Szabo et al., 1993; Rooney et al., 1994; for review, see
Rao(1994)). Induction of NFAT parallels that of IL-2 and requires
protein kinase C-dependent and Ca
-dependent signaling
processes that can be triggered in vitro by the combination of
phorbol ester (PMA) and ionomycin or PMA and phytohemagglutinin (PHA)
(Durand et al., 1988; Ullman et al., 1990; Flanagan et al., 1991).
The structural complexity of NFAT was first
appreciated by Flanagan et al.(1991), who proposed a
two-component structure consisting of a PMA-inducible nuclear component
resistant to CsA and FK-506 and a T cell-specific cytosolic component
present in unstimulated T cells that translocated to the nucleus upon
ionomycin or PHA stimulation. The translocation of the cytosolic
component is sensitive to CsA and FK-506, indicating that this step is
controlled directly or indirectly by CaN. Thus, the simplest proposal
to account for activation has been that CaN ``activates''
NFAT solely by controlling translocation of the cytosolic component to
the nucleus (Schreiber and Crabtree, 1992). The PMA-inducible nuclear
component was later shown to consist of ubiquitous Fos and Jun family
proteins (Jain et al., 1992, 1993a; Boise et al.,
1993; Northrop et al., 1993; Yaseen et al., 1993,
1994; Jain et al., 1994). Recently, a pre-existing component
of NFAT (NFATp) (for review, see Rao(1994)) was purified from
cytoplasmic extracts of a murine T cell line (Jain et al.,
1993a), and its cDNA was cloned (McCaffrey et al., 1993b).
Although its expression is not restricted to T cells, its biochemical
characteristics are very similar to the cytosolic component that has
been described by Flanagan et al.(1991). NFATp is present in
the cytoplasm of unstimulated murine T cells, it translocates to the
nucleus upon Ca-dependent stimulation, and its
translocation is blocked by CsA. (
)Purified NFATp is able to
directly bind to the murine IL-2 NFAT motif and to form an NFAT complex
in association with Fos and Jun proteins (Jain et al., 1993a;
McCaffrey et al., 1993b). NFATp is a phosphoprotein with a
molecular mass of
120 kDa that acts as an in vitro substrate of CaN (Jain et al., 1993a; McCaffrey et
al., 1993a), supporting the notion that NFATp is a potential
target of CsA. A related cytoplasmic protein, NFATc, has been
characterized in human T cells. Expression of NFATc in nonlymphoid
cells is able to activate NFAT-driven transcription with PMA and
ionomycin stimulation as well as confer DNA-binding activity (Northrop et al., 1994). The DNA-binding domains of both NFATp and NFATc
display a distinct sequence similarity with the Rel homology region of
Rel proteins (McCaffrey et al., 1993b; Northrop et
al., 1994). However, whereas NFATp is constitutively expressed in
a variety of lymphoid cells and in certain nonlymphoid cells (Ho et
al., 1994; Wang et al., 1995),
RNA expression
of NFATc is induced by PMA and ionomycin stimulation, partially
inhibited by CsA, and largely restricted to T cells (Northrop et
al., 1994). Furthermore, it has recently been shown that NFAT is
constitutively nuclear in double-positive thymocytes (Sen et
al., 1994). Therefore, it appears that T cell-specific,
CsA-sensitive NFAT-mediated transcription is a complex phenomenon
involving more than one protein regulated by more than one mechanism.
We have recently shown that NFAT is inducible in human B cells upon PMA/PHA stimulation and that a constitutively expressed component present in nuclear extracts of unstimulated transformed B and T cell lines is necessary and sufficient for reconstituting a specific NFAT complex in association with recombinant Fos and Jun proteins (Yaseen et al., 1993, 1994). The constitutive component is therefore similar to NFATp in its ability to form an NFAT complex in combination with Fos and Jun proteins. However, its presence in nuclear extracts of unstimulated human B and T cell lines raised the question of whether and how CsA inhibits the NFAT activity in this system, since CsA has been assumed to inhibit NFAT complex formation by blocking the migration of a cytoplasmic component to the nucleus (Flanagan et al., 1991; Schreiber and Crabtree, 1992). In this report, we show that the constitutive factor found in nuclear extracts of unstimulated B and T cell lines is a human homologue of murine NFATp and that its ability to reconstitute an NFAT complex in cooperation with Fos and Jun is inhibited by CsA treatment. This inhibition is due to loss of DNA-binding activity of human NFATp resulting from an increase in its phosphorylation level as in vitro phosphatase treatment completely restores DNA-binding activity of the factor. These observations demonstrate an additional mechanism for CsA-mediated inhibition of NFAT complex formation.
Nuclear pellets were
washed once with 1 ml of the same buffer, and resuspended in
300-400 µl of a nuclear extraction buffer containing 20
mM Hepes, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl, 25% (v/v) glycerol, 0.2 mM EDTA, 0.5
mM dithiothreitol, and the above protease inhibitors.
Resuspended nuclei were incubated on ice for 30 min with occasional
shaking to extract the nuclear proteins and finally spun down in a
microcentrifuge for 5 min. The supernatant with nuclear proteins was
dialyzed against a dialysis buffer (20 mM Hepes, pH 7.9, 100
mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol).
For preparation of cytoplasmic fractions, the
300 g supernatant was further centrifuged at 100,000
g for 1 h. Cytoplasmic proteins in the supernatant
were precipitated at 1.5 M ammonium sulfate for 30 min on ice,
and the precipitated proteins were collected by centrifugation at
100,000
g for 30 min. The protein pellets were
resuspended in the dialysis buffer supplemented with the above protease
inhibitors, and dialyzed extensively against the dialysis buffer. The
protein concentration was determined using the Bio-Rad protein assay
kit with bovine serum albumin as a standard.
Figure 4:
In vitro dephosphorylation of
NFATp by calf intestinal AP and CaN. A and B, nuclear
extracts (40 µg) from either CsA-untreated (lanes
1-3) or treated (lanes 4-8) Raji and Jurkat
cells were incubated either alone or with AP or CaN in the presence or
absence of phosphatase inhibitors (I) consisting of 20 mM sodium pyrophosphate and 50 mM NaF, and analyzed in
immunoblot assays using an antiserum to recombinant murine NFATp. Lane8 in panel A is heat-inactivated CaN,
and lane 9 in panel A is nuclear extract of HeLa
cells. NFATp is indicated by arrows. C,
immunoprecipitation of NFATp from nuclear extracts of metabolically P-labeled Raji cells. Raji cells were treated with or
without 1 µg/ml CsA for 2 h in medium containing
[
P]orthophosphate, and nuclear extracts were
prepared as described under ``Experimental Procedures.''
Nuclear extracts (300 µg) from CsA-treated cells were treated with
CaN. NFATp was immunoprecipitated with R59 antiserum from nuclear
extracts and analyzed in an immunoblot assay (left panel). The
same membrane was scanned by a PhosphorImager (right panel). Filled arrow indicates phosphorylated NFATp, and open
arrow indicates dephosphorylated form. D, nuclear
extracts (40 µg) from CsA-treated Raji cells were incubated either
alone (lanes 2 and 7) or with increasing amounts of
CaN (lanes 3-6) or AP (lanes 8-11), and
analyzed in an immunoblot assay. Lane 1 is CsA-untreated. The
amounts of CaN used in each lane were as follows: lane 3, 0.06
µg; lane 4, 0.12 µg; lane 5, 0.25 µg; lane 6, 0.50 µg. Those of AP were as follows: lane
8, 1.4 units; lane 9, 2.8 units; lane 10, 5.6
units; lane 11, 11.2 units. NFATp is indicated by arrows.
For immunoblotting of H subunit of lactate dehydrogenase (LDH) and transcription factor USF, equal amounts of nuclear and cytoplasmic extracts were fractionated on a 10% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The rest of the procedure was the same as that used for NFATp immunoblotting except for the first and second antibodies used. A monoclonal mouse anti-human LDH antibody (Sigma) and 1:2000-diluted goat anti-mouse IgG antibodies conjugated to horseradish peroxidase (Santa Cruz Biotech.) were used for first and second antibody incubations, respectively. After detecting LDH by ECL system, horseradish peroxidase on the filter was inactivated by incubation in Tris-buffered saline containing 0.2% Tween 20 and 15% hydrogen peroxide at room temperature for 30 min. The filter was reprobed with 0.2 µg/ml of rabbit anti-USF antibodies (Santa Cruz Biotech.), and a donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase was used as a secondary antibody.
Figure 1: CsA inhibits DNA binding activity of the constitutive component of NFAT in human lymphocytes. A, inhibition of inducible NFAT complex in T and B cells by CsA treatment. EMSA using a radioactive probe containing the human IL-2 NFAT site was performed with nuclear extracts from Jurkat (a T cell line) and Raji (a B cell line) stimulated with 50 ng/ml PMA and 2 µg/ml PHA in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 µg/ml CsA. NFAT induction was inhibited in both B and T cells by CsA treatment. Arrows indicate NFAT complexes and free probe. B, EMSA was performed using nuclear extracts from unstimulated Raji B and Jurkat T cells, either untreated (lanes 1, 2, 5, and 6) or treated (lanes 3, 4, 7, and 8) with 1 µg/ml of CsA. Extracts were incubated with a labeled human IL-2 NFAT oligonucleotide in the absence (odd-numbered lanes) or presence (even-numbered lanes) of 125 nM recombinant Fos/Jun heterodimers.
We have previously demonstrated that a constitutive component present in nuclear extracts of unstimulated human B and T cell lines could reconstitute an NFAT complex in association with purified recombinant Fos and Jun proteins (Yaseen et al., 1993, 1994). Since Fos and Jun induction is not sensitive to CsA, it was likely that the target for CsA inhibition in NFAT was the constitutive component found in unstimulated B and T cells. To find out whether the constitutive component is a target of CsA, nuclear extracts from unstimulated Raji B and Jurkat T cells treated with or without CsA were tested for their ability to cooperate with recombinant Fos/Jun heterodimers in NFAT reconstitution (Fig. 1B). As expected, nuclear extracts of CsA-untreated B and T cells were able to reconstitute an NFAT complex (lanes 2 and 6). On the other hand, those of CsA-treated cells lost most of their ability to reconstitute an NFAT complex (lanes 4 and 8). These results indicate that the constitutive component of NFAT is a target of CsA, but they do not show whether CsA treatment leads to loss of the component from lymphocyte nuclei or to its modification in a way that prevents it from binding to DNA.
Figure 2: The constitutive component is a human homologue of NFATp. EMSA was performed with nuclear extracts (10 µg) from either PHA/PMA-stimulated (lanes 1-4) or unstimulated (lanes 5-8) Raji cells, using labeled human IL-2 NFAT oligonucleotide as a probe in the presence of preimmune serum, antiserum against recombinant murine NFATp (Ab1), or antiserum against a peptide in murine NFATp (Ab2) as described (see ``Experimental Procedures''). With unstimulated nuclear extracts, 125 nM Fos/Jun heterodimers were added to the reaction mixtures for NFAT reconstitution (lanes 5-8). Since antisera were diluted in phosphate-buffered saline, 1 µl of phosphate-buffered saline was added to the control reactions (lanes 1 and 5). Native and reconstituted NFAT complexes are indicated by an arrow.
Figure 3: Effect of CsA on posttranslational modification of NFATp. A, immunoblot analysis of NFATp was performed with both nuclear and cytoplasmic extracts. Nuclear and ammonium sulfate-precipitated cytoplasmic proteins were prepared in equal volumes from Raji and Jurkat cells either untreated or treated with 1 µg/ml CsA as described under ``Experimental Procedures.'' Equal volumes of nuclear (N) and cytoplasmic (C) extracts were separated on 7% SDS-polyacrylamide gels, transferred on nitrocellulose membranes, and immunoblotted for NFATp with an antiserum to recombinant murine NFATp. The amounts of proteins loaded on each lane were as follows: lane 1, 30 µg; lane 2, 71 µg; lane 3, 31 µg; lane 4, 100 µg; lane 5, 30 µg; lane 6, 47 µg; lane 7, 28 µg; lane 8, 39 µg. Arrows indicate NFATp, and molecular mass standards (in kDa) are marked at the right.B, immunoblot analyses performed with antibodies raised against a nuclear protein (USF) and a cytoplasmic protein (LDH) demonstrate absence of significant contamination of nuclear extracts with cytoplasmic proteins. Equal volumes of the nuclear (N) and cytoplasmic (C) extracts used in panel A were separated on a 10% SDS-polyacrylamide gel and analyzed in immunoblot assays using either anti-USF antibody (top) or anti-LDH antibody (bottom).
Since NFATp has been reported to be mainly a cytoplasmic protein in unstimulated murine T cells (McCaffrey et al., 1993a), it was important to rule out significant contamination of nuclear extracts with cytoplasmic proteins. Nuclear and cytoplasmic extracts prepared by the same method were tested in immunoblot analyses with antibodies against a cytoplasmic protein marker, the LDH (Cahn et al., 1962), or to a nuclear protein marker, USF. The USF transcription factor, composed of 43- and 44-kDa polypeptides, is a member of the basic, helix-loop-helix family and localized predominantly in the nucleus (Sawadogo et al., 1988; Pognonec and Roeder, 1991). The majority of USF was detected in nuclear extracts prepared from both B and T cells (Fig. 3B, top), whereas LDH was mainly observed in cytoplasmic extracts (Fig. 3B, bottom). These data rule out any significant contamination of nuclear extracts with cytoplasmic proteins.
To further confirm that the migration shifts observed with CsA and
CaN treatments are indeed due to changes in the phosphorylation level
of NFATp, Raji cells were metabolically labeled with
[P]orthophosphate in the presence or absence of
CsA treatment. Nuclear extracts were treated with or without CaN, and
NFATp was immunoprecipitated, separated on SDS-polyacrylamide gel
electrophoresis, and transferred to nitrocellulose membrane (Fig. 4C). Immunoblotting with NFATp antibody confirmed
the migration shift and the presence of similar amounts of NFATp in the
different samples (Fig. 4C, left panel).
PhosphorImager scan of the same membrane showed that CsA treatment
resulted in an increased level of phosphorylation of NFATp and that the
phosphorylation level returned to base line after CaN treatment of
nuclear extract (Fig. 4C, right panel). These
data confirm that NFATp migration shifts in response to CsA and
phosphatase treatments are accompanied by appropriate shifts in its
phosphorylation level.
The smaller migration shift after AP treatment suggests that AP dephosphorylates fewer sites than CaN. Furthermore, comigration of Jurkat and Raji NFATp after CaN treatment suggests that NFATp in Jurkat cells may be identical to that of Raji cells and that the difference in migration observed without phosphatase treatments (Fig. 3A, lanes 1 and 5) was due either to different degrees of phosphorylation of NFATp in B versus T cells or to differential nonspecific phosphatase activity during extract preparation. To exclude the possibility that the differential effects of AP and CaN on NFATp migration are due to less than optimal enzyme concentrations, nuclear extracts from CsA-treated Raji cells were treated with increasing amounts of CaN or AP and analyzed in an immunoblot assay (Fig. 4D). The faster migrating form of NFATp began to appear at 0.12 µg of CaN treatment (lane 4) and at 0.25 µg of CaN all NFATp became faster migrating (lane 5); further migration shift was not even observed up to 2 µg of CaN treatment as shown earlier in Fig. 4A. Similarly, NFATp migration was faster, but not as much as that of CaN treatment, at 1 unit of AP, and a higher concentration of AP up to 12 units did not generate further migration shift. Thus, a smaller migration shift observed with AP as compared with that observed with CaN is not due to suboptimal enzyme concentration and is most likely due to dephosphorylation of fewer sites.
Figure 5: Dephosphorylation of NFATp restores reconstitution of NFAT complex in nuclear extracts from CsA-treated cells. A, nuclear extracts from CsA-untreated or treated Raji (top panel) and Jurkat (bottom panel) cells were treated with (lanes 5-8) or without (lanes 1-4) AP, and subsequently tested for DNA-binding to a human IL-2 NFAT oligonucleotide in the presence (even numbered lanes) or absence (odd numbered lanes) of Fos/Jun heterodimers. B, same experiment as in A but with CaN instead of AP. In addition to CaN, 50 mM NaF and 20 mM sodium pyrophosphate were added to the reaction mixture to inhibit CaN phosphatase activity in lane9, bottompanel. Reconstituted NFAT complexes are indicated by an arrow. The faint band observed under the reconstituted NFAT complex represents weak direct binding of Fos/Jun heterodimers to the human IL-2 NFAT site (Yaseen et al., 1994).
Minor migration differences observed in NFAT complexes restored by phosphatase treatment (Fig. 5A, top panel, compare lane 2 with lane 8, and Fig. 5B, bottom panel, compare lane 2 with lanes 6 and 8) are probably due to differing degrees of dephosphorylation of NFATp under the conditions indicated, which also resulted in different migrations on SDS-polyacrylamide gels (Fig. 4).
Figure 6: In vitro dephosphorylation of human NFATp restores direct binding to the murine NFAT site. Nuclear extracts from CsA-treated Raji cells were incubated either alone (lane 2) or with AP (lanes 3 and 5) or CaN (lanes 4 and 6) in the presence or absence of phosphatase inhibitors (I) and tested for DNA-binding activity to a murine IL-2 NFAT oligonucleotide in EMSA. EMSA was performed with a nuclear extract from CsA-untreated Raji cells for comparison (lane 1). Sodium pyrophosphate (20 mM) and NaF (50 mM) were added to the phosphatase reaction mixtures to inhibit phosphatase activity of AP (lane 5) and CaN (lane 6). NFATp-DNA complexes are indicated by arrows.
To determine whether dephosphorylation of
NFATp can restore its direct DNA-binding activity, nuclear extracts
from CsA-treated Raji B cells were treated with either AP or CaN and
analyzed for binding to the murine IL-2 NFAT motif by EMSA. DNA-binding
activity of NFATp in CsA-treated cells was restored by AP and CaN
treatments (lanes 4 and 5, respectively), and the
restored DNA binding was specific to the murine NFAT motif, based on
unlabeled oligonucleotide competition and supershifts with antisera to
murine NFATp (data not shown). Phosphatase inhibitors blocked recovery
of DNA binding of NFATp to a murine IL-2 NFAT oligonucleotide,
indicating that restoration resulted from dephosphorylation of NFATp (lanes 5 and 6). The NFATpDNA complex restored
by AP migrated slower than that of CsA-untreated cells, while the
complex restored by CaN migrated at a position identical to that of CsA
untreated cells. Together with the apparent molecular weight
differences in immunoblot analyses generated by AP and CaN treatments
of nuclear extracts (Fig. 4A, lanes 5 and 6), this indicates differing degrees of dephosphorylation of
NFATp and suggests that only a subset of phosphorylated sites is
involved in inhibiting DNA binding.
A widely accepted model of CsA-mediated inhibition of
cytokine gene expression involves inactivation of a calcium-dependent
phosphatase and sequestration of a cytoplasmic component(s) of the
transcription factor NFAT. It has been suggested that in activated T
cells, a cytoplasmic component(s) is dephosphorylated by the
Ca-dependent phosphatase CaN resulting in its
translocation to the nucleus where it joins Fos/Jun proteins to form
the NFAT complex (Clipstone and Crabtree, 1992; McCaffrey et
al., 1993a). However, the fact that unstimulated human Raji B and
Jurkat T cell lines constitutively express a nuclear component that can
participate in reconstitution of the NFAT complex in the presence of
Fos/Jun heterodimers (Yaseen et al., 1993, 1994) poses the
question of whether this nuclear non-AP-1 component is also a target
for CsA in the absence of a calcium signal. Our preliminary experiments
showed that CsA treatment of unstimulated Raji and Jurkat cells results
in loss of reconstitution of the NFAT complex in the presence of
Fos/Jun heterodimers (Fig. 1B). This observation cannot
be accounted for by the loss of stimulation-dependent translocation of
a cytoplasmic component(s) to the nucleus, since it occurred in
unstimulated cells in which the factor is constitutively nuclear. The
data presented here show that this factor is a human homologue of
murine NFATp and point to an additional mechanism for the CsA-mediated
inhibition of NFAT activity in T and B cells.
Several lines of
evidence suggest that the constitutive component that we detect in
nuclear extracts of unstimulated B and T cell lines is a human
counterpart of murine NFATp. 1) Both proteins are able to reconstitute
an NFAT complex with murine and human IL-2 NFAT binding sites in the
presence of Fos/Jun heterodimers; both also bind specifically to a
murine NFAT oligonucleotide without Fos and Jun proteins (Jain et
al., 1992, 1993b; Yaseen et al., 1993; Fig. 1and Fig. 6). 2) Both are phosphoproteins with an apparent molecular
mass of 120 kDa and serve as in vitro substrates of CaN
(McCaffrey et al., 1993a; Fig. 3and Fig. 4). 3)
Both are targets of CsA (McCaffrey et al., 1993a; this work).
4) Antisera specific to the murine NFATp (McCaffrey et al.,
1993b; Ho et al., 1994) supershift NFAT complexes formed by
the human constitutive component on both human and murine NFAT
oligonucleotides (Fig. 2) and recognize it on immunoblots ( Fig. 3and 4). On the other hand, the observation that an NFATp
homologue is constitutively nuclear in transformed human B and T cell
lines is intriguing as NFATp is mainly cytoplasmic in unstimulated
murine T cells (McCaffrey et al., 1993a). However, these two
observations are not mutually exclusive. One interpretation of this
difference is that transformation-associated activation in Raji B and
Jurkat T cells may result in nuclear localization of NFATp.
The DNA
binding activity of nuclear NFATp in unstimulated Raji and Jurkat cells
was strongly inhibited by CsA treatment ( Fig. 1and Fig. 6), an effect that could be observed within the first hour
of treatment (data not shown). The loss of DNA-binding activity was due
to increased phosphorylation of NFATp as in vitro treatment of
nuclear extracts from CsA-treated cells with AP and CaN could restore
this activity ( Fig. 5and Fig. 6). Increased
phosphorylation of NFATp appears to be the main consequence of
CsA-treatment in Raji and Jurkat cell lines, with no appreciable change
in its subcellular distribution ( Fig. 3and 4). Thus, CsA not
only inhibits the translocation of NFATp to the nucleus, but also
inhibits the DNA binding activity of NFATp that is already in the
nucleus. These observations imply that CsA can prevent NFAT-mediated
transactivation even when NFATp is already in the nucleus as is the
case in transformed cells. Indeed, CsA can inhibit NFATp DNA binding
activity when added up to 4 h after in vitro cell stimulation
by PHA and PMA. ()
It is interesting to note that AP
dephosphorylation of CsA-treated extracts resulted in a smaller
migration shift on a SDS-polyacrylamide gel than CaN dephosphorylation,
although both treatments resulted in complete restoration of DNA
binding activity (Fig. 4). Furthermore, NFATp from Jurkat and
Raji cells shows a slight difference in migration on SDS-polyacrylamide
gel electrophoresis (Fig. 3). This difference is reduced by AP
treatment and completely abolished by CaN treatment (Fig. 4).
These findings suggest that there are partially dephosphorylated forms
of NFATp in Raji and Jurkat cells, and this dephosphorylation may
involve CaN or other phosphatases. Indeed, treatment of Jurkat cells
with ionomycin led to further dephosphorylation of NFATp, which may be
necessary for transcriptional activation but not for DNA-binding. Consequently, NFATp may have multiple phosphorylation sites, only
some of which are involved in the regulation of DNA-binding activity,
while others may subserve different functions such as transcriptional
activation and subcellular localization.
In summary, our findings demonstrate that immunosuppressive drugs can inhibit the activation of NFAT by directly preventing the DNA binding ability of NFATp through increased phosphorylation, suggesting that these drugs can affect NFAT function at multiple steps. Our data also suggest the presence of multiple physiologically important phosphorylation sites within the NFAT molecule and raise the question of whether CaN is the only phosphatase involved in NFAT regulation and in the response to CsA and FK-506.