From the Institute of Medical Microbiology and
Immunology and § Institute for Medical Biochemistry and
Genetics, University of Copenhagen and the ¶ Institute for
inflammation Research, Rigshospitalet and
Department of Clinical
Immunology, National University Hospital, DK2200 Copenhagen,
Denmark
Received for publication, October 4, 2002
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ABSTRACT |
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Interleukin-4 (IL-4) plays a pivotal role in the
induction and maintenance of allergy by promoting Th2 differentiation
and B cell isotype switching to IgE. Studies on STAT6-deficient mice have demonstrated the essential role of STAT6 in mediating the biological functions of IL-4. IL-4 induces tyrosine phosphorylation of
STAT6, which in turn leads to transcription of IL-4-specific genes. In
addition, serine phosphorylation of STAT6 has recently been reported.
Here we study the functional role of STAT6 serine phosphorylation and
the kinases and phosphatases involved. We show that inhibition of
protein phosphatase 2A (PP2A) induces serine phosphorylation of STAT6
and severely inhibits DNA binding of STAT6. In contrast, IL-4-induced
tyrosine phosphorylation of Janus kinase-1 and STAT6 is not affected,
suggesting that PP2A acts downstream of Janus kinases in IL-4
signaling. In conclusion, we provide the first evidence that PP2A plays
a crucial role in the regulation of STAT6 function.
IL-41 is an important
cytokine, which regulates the growth, differentiation, and survival of
a variety of cell types. Thus, IL-4 plays a key role in the
differentiation of native CD4+ T cells into Th2 T cells and
in the regulation of apoptosis and growth of B cells. Furthermore, IL-4
induces B cell immunoglobulin isotype switching to IgG1 and
IgE (reviewed in Refs. 1 and 2). Stimulation of the IL-4 receptor
complex by IL-4 results in the activation of multiple signaling
pathways, one of which involves signal transducers and activators of
transcription-6 (STAT6). IL-4 induces rapid tyrosine phosphorylation of
STAT6 by IL-4 receptor-associated Janus kinases (JAK-1 and JAK-3),
which in turn leads to STAT6 dimerization and rapid translocation to the nucleus, where STAT6 acts as an activator of IL-4-specific gene
transcription (reviewed in Refs. 2 and 3). Studies of STAT6-deficient
mice have shown that STAT6 plays a critical role in IL-4 signaling and
the induction of allergy (4, 5). Recent reports suggest that IL-4, in
addition to tyrosine phosphorylation, also induces serine
phosphorylation of STAT6, but it is not known which role serine
phosphorylation plays in the regulation of STAT6 signaling and which
kinases and phosphatases are involved (6, 7).
At present four major classes of serine/threonine-specific protein
phosphatases (PPases) are known. These include two that are
Ca2+-independent (PP1 and PP2A) and two that are
Ca2+-dependent (PP2B (calcineurin) and PP2C).
PP1 and PP2A are expressed ubiquitously in eukaryotic cells and are
reportedly involved in several signaling pathways (reviewed in Refs. 8
and 9). Here, we provide the first evidence that PP2A regulates
IL-4-mediated STAT6 signaling.
Cell Lines and Plasmid Construct--
Antigen-specific human
CD4+ T cell lines were obtained from healthy donors and
have been described elsewhere (10). The cutaneous tumor T cell line
MF2000 has been described (11). Jurkat T cell line J76.25.20 and J-TAg
have been described previously (12). The STAT6 reporter construct
(ST6-pGL3) driving the firefly luciferase gene was made by ligating
pGL3 basic (Promega, Madison, WI) with an oligonucleotide (sense,
5'-CCGACTTCCCAAGAACGTGCTTCCCAAGAACTCTCTTCCCAAGAACAGATCTGGG-TATATAATGGAAGC-3'; antisense,
5'-TCGAGCTTCCATTATATACCCAGATCTGTTC-TTGGGAAGAGAGTTCTTGGGAAGCACGTTCTTGGGAAGTCGGGTAC-3') that contains three copies of the STAT6 binding sequence from the IgE
promotor (IgE3(5), Ref. 13). This IgE promoter sequence was
fused with the minimal TA promotor and then flanked by a
KpnI and a XhoI restriction site. The wild-type
STAT6 expression vector (TPU388), a kind gift from Dr. Ulrike
Schindler, has been described previously (14).
Antibodies and Other Reagents--
Antibodies against human
Stat6 (C-20, sc-621) and Stat4 (C-20, sc-486) were from Santa Cruz
Biotechnology (Santa Cruz, CA), antibodies against human pY
Stat6 (9361) were purchased from New England Biolabs (Beverly, MA),
antibody against human pY Stat3 (stat3-9E12) was from nanoTools
Antikörpertechnik (Denzlingen, Germany), antibody against human
pY JAK-1 was from BIOSOURCE (Carmarillo, CA), and antibodies against human Jak-1 (J24320) and Stat3 (S21320) were from Transduction Laboratories (Lexington, KY). Human
recombinant IL-4 (I-186) was from Leinco Technologies (St. Louis, MO).
Purified PP1 (14-110) and PP2A (14-111) were from Upstate
Biotechnology (Lake Placid, NY), and calf intestine alkaline
phosphatase was from Sigma-Aldrich. Calyculin A, endothall
thioanhydride, tautomycin, okadaic acid, staurosporine, SB203580,
PD169316, wortmannin, LY294002, and casein II kinase inhibitor
(5,6-dichloro-1- Phosphatase Assay--
Antigen-specific human CD4+ T
cells (106/100 µl) were incubated with or without the
PP1/PP2A inhibitor, calyculin A, for 30 min at 37 °C. The activity
of PP1/PP2A in vivo was tested as described previously (15).
Percentage inhibition of PP1/PP2A activity by calyculin A was
calculated according to the formula: percentage inhibition = [(cpm Protein Extraction and Western Blotting--
Following
incubation in medium with or without inhibitors and stimulation with
rhIL-4 the cells were pelleted rapidly, and the reaction was stopped by
lysing the cells in ice-cold lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10%
glycerol, with the following inhibitors: 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 4 µM iodoacetamide, 5 mM EDTA, 10 mM NaF, 10 µg/ml aprotinin). SDS sample buffer was added
to the lysates, which were subsequently subjected to 10%
SDS-PAGE and transferred onto nitrocellulose membranes. Preparations of cytoplasmic/nuclear extracts were conducted as described previously (16). Blots were evaluated by using enhanced chemiluminescence, stripped, and reprobed according to the manufacturer's manual (Amersham Pharmacia Biotech).
Oligonucleotide Affinity Purification of STAT6--
Cytoplasmic
extracts from T lymphoma cells were prepared as described above.
Lysates were precleared with streptavidin-coated agarose beads
(KEM-EN-TEC, Copenhagen, Denmark) and incubated with biotinylated
double-stranded STAT6 binding sequence from the IgE promoter (IgE3(5),
5'-Bio-CGACTTCCCAAGAACGT-GCTTCCCAAGAACTCTCTTCCCAAGAAC-3') (13). STAT-DNA complexes were precipitated using streptavidin-coated agarose beads. Purified DNA-binding proteins were boiled in SDS sample
buffer and analyzed by SDS-PAGE and Western blotting.
In Vitro Dephosphorylation--
Cells were incubated with or
without calyculin A for 60 min prior to lysis in buffer (1% Nonidet
P-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10%
glycerol, without inhibitors). The lysates were left on ice for 20 min,
and thereafter the insoluble parts were removed (13,000 × g, 10 min). Then, the lysates were incubated with or without
0.5 unit/ml purified PP1 or PP2A at 37 °C for 120 min. The reactions
were stopped by adding SDS sample buffer and boiling the samples for 5 min.
Thin-layer Electrophoresis of Phosphoamino Acids--
Thin-layer
electrophoresis was conducted as described previously (17). Briefly,
cutaneous tumor T cells (40 × 106 cells/sample)
radiolabeled with [32P]orthophosphate and
32P-labeled STAT6 were immunoprecipitated before
immunocomplexes were boiled and separated by SDS-PAGE. Pre-stained
standards (Novex, San Diego) were used as molecular weight markers.
Resolved profiles and molecular weight markers were transferred
electrophoretically from SDS polyacrylamide gel onto a
polyvinylidene difluoride membrane, and subjected to autoradiography
overnight without a screen. 32P-Labeled proteins of
interest were cut out from the polyvinylidene difluoride membrane and
incubated in 6 M HCl for 2 min at 100 °C. The samples
were then vortexed followed by hydrolysis at 110 °C for 60 min and
dried with a flow of nitrogen. The samples were then run on 20 × 20-cm cellulose k-2F plates (60 mA, 55 min) in the presence of
o-phosphoamino acid markers. The markers were visualized by
spraying the plate with ninhydrin and o-phtalaldehyde in UV
light. The labeled phosphoamino acids were detected by autoradiography.
Reporter Assay--
In cotransfection experiments 3 × 106 Jurkat J-TAg cells were transfected with 1 µg of
ST6-pGL3 reporter construct, 2 µg of wild-type STAT6 expression
vector (TPU388), 1 µg of internal control pCMV-LacZ, and 4 µl of
DMRIE-C (Invitrogen). Cells were rested for 24 h before being
pretreated without or with calyculin A (20 or 40 nM) for
1 h prior to stimulation with IL-4 (25 ng/ml) for 18 h.
Luciferase and To measure the effect of calyculin A on phosphatase activity in
intact cells, T cell lines were incubated with calyculin A for 60 min
at 37 °C in a humidified atmosphere and washed extensively prior to
analysis for PP1/PP2A activity. As shown in Fig.
1A, calyculin A induced a
concentration-dependent inhibition of PP1/PP2A activity. In
contrast, the PP2B inhibitor, cyclosporin A, had no effect on PP1/PP2A
activity even at concentrations that blocked CD3
antibody-induced proliferation (Ref. 18; data not shown). To
investigate whether PP1 and/or PP2A are involved in the regulation of
STAT6, T cells were incubated with phosphatase inhibitors as described
above, and total cell lysates were subsequently analyzed by Western
blotting using an anti-STAT6 antibody. As shown in Fig. 1B
(lane 5), incubation with calyculin A induced a change in
the electrophoretic mobility of STAT6. Similar results were obtained
with other PP2A inhibitors (okadaic acid, endothall thioanhydride, Fig.
1B, lanes 3-4) but not with inhibitors of PP1
(Tau, Fig. 1B, lane 2) and PP2B
(CyA, Fig. 1B, lane 6). Similar
effects of calyculin A on STAT6 electrophoretic mobility were observed
in antigen-specific CD4+ T cell lines and T cell
lymphoma/leukemia cell lines, indicating that the effect of PP2A
inhibition is not limited to a specific cell line (data not shown).
Phosphoamino acid analysis of immunoprecipitated STAT6 showed that
calyculin A induces a strong increase of serine phosphorylation of
STAT6 (Fig. 1C), which is compatible with the induced change
in electrophoretic mobility of STAT6 (Fig. 1B). In contrast
to the strong effect on serine phosphorylation, threonine phosphorylation was barely detectable following calyculin A treatment, indicating that calyculin A selectively modulates serine
phosphorylation of STAT6.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosyl benzimidasol)
were purchased from Alexis (Läufel-Fingen, Switzerland). PD98059
was from New England Biolabs, and cyclosporin A was from Sigma-Aldrich.
blank)
(cal A cpm
blank) × 100%/(cpm
blank)] where "cal A cpm" indicates the cpm of
the test sample with calyculin A, "blank" indicates the cpm
obtained from the sample without cell lysate, and "cpm" indicates
cpm of the sample without calyculin A. PP1/PP2A activity index 100 was
set at "percentage inhibition" in the sample without calyculin A.
-galactosidase activities were assayed accordingly to
the instructions of the Promega luciferase assay system, and the
Invitrogen
-galactosidase assay kit, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, calyculin A inhibits the activity of
PP1/PP2A in vivo. Resting, antigen-specific, human
CD4+ T cells were incubated with increasing concentrations
of calyculin A for 30 min, and thereafter the cells were pelleted and
lysed in lysis buffer without phosphatase inhibitors. The PP1/PP2A
activity was measured as described under "Experimental Procedures."
B, PP2A- but not PP1-specific inhibitors induced a mobility
shift of STAT6. T lymphoma cells were incubated in tautomycin
(Tau, 500 nM), okadaic acid (OA, 500 nM), endothall thioanhydride (ETA, 100 µM), calyculin A (CA, 80 nM), or
cyclosporin A (CyA, 400 ng/ml) for 60 min. Cells were lysed,
applied to SDS-PAGE as described under "Experimental Procedures,"
and immunoblotted with anti-STAT6. C, amino acid analysis of
32P-labeled STAT6. 32P-labeled proteins of
interest, representing STAT6 proteins, were immunoprecipitated with
anti-STAT6 polyclonal antibody from cutaneous T lymphoma cells
incubated with or without calyculin A for 60 min and hydrolyzed in 6 M HCl before separation by thin-layer electrophoresis. The
32P-labeled phosphoamino acids were detected by
autoradiography as described under "Experimental Procedures."
D, T lymphoma cells (lanes 1-8) or Jurkat T
cells (lanes 9-12) were incubated in medium or calyculin A
(80 nM) for 60 min before cells were lysed in lysis
buffer without inhibitors. Lysates were treated thereafter with
nothing ( ), purified PP2A, or PP1 for 120 min before
subjection to Western blotting with anti-STAT6 polyclonal
antibody.
To further investigate the involvement of PP2A in the regulation of STAT6 phosphorylation, we studied the effect of purified phosphatases in vitro. Total cell lysates from cutaneous T cells (Fig. 1D, lanes 1-8) or Jurkat T cells (Fig. 1D, lanes 9-12) pretreated with or without calyculin A were incubated with purified PP1 or PP2A enzyme prior to analysis by Western blotting with anti-STAT6 antibody. As showed in Fig. 1D, purified PP2A almost completely blocked the calyculin A-induced shift in electrophoretic mobility of STAT6 (lane 4 versus 2 and lane 10 versus 12). In parallel experiments purified PP2A enzyme had no effect on IL-4-induced tyrosine phosphorylation of STAT6 in either cytoplasmic or nuclear extracts (data not shown). In contrast, purified PP1 had no effect on the calyculin A-induced mobility shift of STAT6 in cutaneous T cells (Fig. 1D, lane 6 versus 2) and Jurkat T cells (data not shown). Taken together these data indicate that inhibition of PP2A triggers serine-phosphorylation of STAT6 in vivo, which can be dephosphorylated by PP2A in vitro.
In an attempt to identify the serine kinase responsible for the serine
phosphorylation of STAT6, T cells were incubated with inhibitors of
candidate serine kinases prior to treatment with calyculin A. Inhibitors of serine/threonine kinases such as MEK (PD098059), p38 MAPK
(SB203580), JNK (PD169316), casein II-kinase (5,6-dichloro-1--D-ribofuranosyl benzimidasol),
phosphatidylinositol 3-kinase (wortmannin, LY294002), and the
broad-spectrum kinase inhibitor H7 had no effect on calyculin A-induced
phosphorylation of STAT6 (Fig. 2,
lanes 3-8 and data not shown). However, preincubation with
staurosporine, a broad-spectrum inhibitor of
serine/threonine-kinases (19), was able to inhibit a calyculin
A-induced mobility shift of STAT6 (Fig. 2, lane 10),
suggesting that an as yet unidentified, staurosporine-sensitive serine
kinase is involved in the regulation of serine phosphorylation of
STAT6.
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Because STAT6 mediates IL-4-induced gene activation and plays a
critical role in cytokine-induced IgE responses, we addressed the
question of whether PP2A regulates IL-4-mediated STAT6 activation. Accordingly, T cells were preincubated with calyculin A prior to
stimulation with IL-4, and total cell lysates were subsequently analyzed by Western blotting using an anti-pY STAT6 antibody. As shown
in Fig. 3A (upper
panel), IL-4 induced a strong tyrosine phosphorylation of STAT6.
Preincubation with calyculin A did not affect the amount of
IL-4-induced tyrosine-phosphorylated STAT6 but triggered a significant
change in the electrophoretic mobility of tyrosine-phosphorylated STAT6
(Fig. 3A, upper panel, lane 4 versus 3). Stripping and reprobing of the membrane with
antibodies directed against total STAT6 and STAT4 showed that calyculin
A induced a similar shift in electrophoretic mobility of STAT6 in IL-4-stimulated (and unstimulated) T cells (Fig. 3A,
middle panel, lanes 4 and 2), and that
the electrophoretic mobility of STAT4 was unaffected by calyculin A
(Fig. 3A, lower panel). As shown in Fig.
3B, preincubation with calyculin A did not have any effect on IL-4-induced JAK-1 activation.
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To address whether PP2A modulates IL-4-induced nuclear translocation of
STAT6, cells were treated with phosphatase inhibitors and analyzed for
STAT6 distribution in the cytosolic and nuclear fractions. As expected,
the PP2A inhibitor calyculin A induced a significant shift in
electrophoretic mobility of cytosolic STAT6, whereas inhibitors of PP1
and PP2B did not (Fig. 4A,
upper panel). The amount of IL-4-induced tyrosine
phosphorylation of STAT6 in the cytosol was unaffected by pretreatment
with protein phosphatase inhibitors (Fig. 4A, upper
panel). In contrast, phosphotyrosine STAT6 was almost completely
absent in nuclear extracts from calyculin A-treated cells (Fig.
4A, lower panel, lane 5 versus
4). Stripping and reblotting with an antibody against total STAT6
showed that STAT6 was present in the nuclear extracts and that IL-4
induced a significant increase in the amount of total STAT6 (Fig.
4A, lower panel, lane 4 versus
1). Calyculin A did not inhibit the IL-4-mediated increase of
total STAT6 in the nuclear fraction (Fig. 4A, lower
panel, lane 5 versus 2) but induced a shift
in the electrophoretic mobility of STAT6 in nuclear extracts that was
comparable (but not identical) to that seen in the cytosolic fraction
(Fig. 4A, lane 5, lower
versus upper panel). In contrast to the
inhibition of phosphotyrosine STAT6 by calyculin A, inhibitors of PP1
and PP2B had no effect on phosphotyrosine STAT6 (and total STAT6) in
nuclear extracts (Fig. 4A, lower panel).
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To address whether calyculin-induced serine phosphorylation influences
the ability of STAT6 to bind promoter regions of IL-4 target genes, we
took advantage of the oligonucleotide "fishing" assay (20). In this
assay, biotinylated oligonucleotide probes representing the
STAT6-binding site from the IgE promoter were used to precipitate
promoter-binding proteins (13). As shown elsewhere (21) and confirmed
in Fig. 4B (lane 3), IL-4 induced a strong
binding of tyrosine-phosphorylated STAT6 from the cytoplasm to
the IgE oligonucleotide sequence. Although calyculin did not inhibit
tyrosine phosphorylation of STAT6 in the cytosol (Fig. 4A),
preincubation with calyculin A almost completely blocked the binding of
tyrosine phosphorylated STAT6 to the oligonucleotide probe (Fig.
4B, lanes 2 and 4), suggesting that
calyculin A-induced serine phosphorylation blocks the DNA binding
function of STAT6. To investigate whether pretreatment with calyculin A
also inhibits the transcriptional activity of IL-4-induced STAT6, we
made a STAT6-responsive luciferase expression construct. As
shown in Fig. 4C luciferase expression induced after 18 h of IL-4 stimulation was completely inhibited by preincubation with
calyculin A (20 and 40 nM).
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DISCUSSION |
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In the present study we provide the first evidence that protein phosphatases are involved in the regulation of STAT6. Inhibition of PP1/PP2A phosphatase activity by calyculin A triggered serine phosphorylation and a shift in the electrophoretic mobility of STAT6. Two structurally different PP2A inhibitors, okadaic acid and endothall thioanhydride, also induced a significant shift in the electrophoretic mobility of STAT6, whereas inhibitors of PP1 (tautomycin) and PP2B (cyclosporin A) did not, suggesting that PP2A rather than PP1 (and PP2B) plays a key role in the regulation of STAT6. This conclusion is in accordance with our observation that treatment of cell lysates with purified PP2A restored the electrophoretic mobility of STAT6, whereas incubation with PP1 enzyme did not. Phosphoamino acid analysis of STAT6 immunoprecipitated from T cells incubated with or without calyculin A showed that inhibition of PP2A leads to a strong increase in serine phosphorylation in vivo. In contrast, calyculin A did not induce threonine phosphorylation, suggesting that PP2A selectively regulates serine phosphorylation of STAT6.
In an attempt to identify the kinase responsible for serine phosphorylation of STAT6, we took advantage of cell-permeable inhibitors of candidate serine kinases. It has recently been shown that calyculin A induces MAPK (ERK1/2) activation through a PD98059-sensitive pathway in T cells (17, 22), and MAP and JNK kinases have been implicated in cytokine-induced serine phosphorylation of STAT1, STAT3, and/or STAT5 (23-25). Therefore, we explored the possible role of MAP and JNK kinase pathways in calyculin A-induced STAT6 phosphorylation. Despite an almost complete inhibition of ERK1/2 activation (data not shown), the MEK inhibitor PD98059 did not inhibit the effect of calyculin A on STAT6, indicating that the serine phosphorylation of STAT6 was not mediated via the MEK/MAPK pathway. In support of this conclusion, STAT6 lacks the PSXP MAPK consensus phosphorylation site found in STAT3 and STAT1 (7), and the PSP motif found in STAT5a and -5b (23). Inhibitors of the P38 and JNK pathways also failed to inhibit the calyculin A-induced mobility shift of STAT6. In contrast, staurosporine inhibited the calyculin A effect on STAT6, suggesting that an as yet unidentified, staurosporine-sensitive kinase was responsible for calyculin A-induced serine phosphorylation of STAT6. This is in agreement with our previous findings that staurosporine blocks serine phosphorylation of STAT3 in response to PP2A inhibitors (17). We therefore hypothesize that the level of serine phosphorylation of STAT6 results from a balance between phosphorylation by a constitutively active, staurosporine-sensitive kinase and dephosphorylation by PP2A. This hypothesis is supported by our finding that STAT6 is a substrate for PP2A in vitro. Alternatively, PP2A may function as a negative regulator of a staurosporine-sensitive kinase.
Our observation that calyculin A did not inhibit IL-4-induced activation of JAK-1 and STAT6 indicates that PP2A is not involved in the initial signaling events following IL-4R ligation. This conclusion was in accordance with our finding that inhibition of PP2A activity did not inhibit IL-4-mediated translocation of STAT6 to the nucleus. Because we were unable to detect tyrosine-phosphorylated STAT6 in the nucleus of IL-4-stimulated cells, it appears that calyculin A treatment induced nuclear tyrosine dephosphorylation of STAT6. Alternatively, calyculin A might induce re-export from the nucleus of tyrosine-phosphorylated STAT6. A more trivial explanation could be that calyculin A, directly or indirectly, interfered with antibody recognition of tyrosine-phosphorylated STAT6 isolated from the nucleus. However, our observation that antibody recognition of tyrosine-phosphorylated STAT6 obtained from the cytosolic fraction was unaffected by calyculin A strongly argues against this possibility.
It has been a matter of some controversy as to how serine phosphorylation modulates the function of STAT proteins. Thus, serine phosphorylation has been reported to increase or decrease the level of tyrosine phosphorylation and/or the transcriptional activity of STAT1, STAT3, and/or STAT5 (22, 26-30). Two recent papers reported that IL-4 induces serine phosphorylation of STAT6 (6, 7), but the authors were not able to ascribe any functional effect of serine phosphorylation on the STAT6 signaling pathway. Here we show that inhibition of PP2A induces serine phosphorylation and inhibition of both the DNA binding capacity and the transcriptional activity of STAT6.
Because a major site for serine phosphorylation lies within the known
transactivation domain of STAT6 (7), it is possible that inhibition of
STAT6 binding to the IgE promoter is caused by calyculin A-mediated
serine phosphorylation of the transactivation domain. Studies are in
progress to address this hypothesis. In conclusion, we have
provided the first evidence that PP2A regulates cytokine-mediated STAT6 signaling.
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FOOTNOTES |
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* This work was supported in part by the University of Copenhagen Ph.D. program (for A. W. and J. B.), The Danish Research Councils, Danish Biotechnological Center for Cellular Communication, Danish Biotechnology Program, Danish Allergy Research Center, Novo Nordic Foundation, Becketts Fond, Danish Medical Associations Research Foundation, the Danish Cancer Research Foundation (Dansk Kræftsforsknings Fond), the Danish Cancer Society (Kræftens Bekæmpelse), the Gerda and Aage Haensch's Foundation, and the Danish Rheumatism Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Institute of Medical Microbiology and Immunology, Panum 22.5.34, University of Copenhagen, Blegdamsvej 3c, DK2200 Copenhagen, Denmark. Tel.: 45-3532-7879; Fax: 45-3532-7863; E-mail: N.Odum@immi.ku.dk.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M210196200
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ABBREVIATIONS |
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The abbreviations used are: IL-4, interleukin-4; STAT, signal transducers and activators of transcription; JAK, Janus kinase; PPase, protein phosphatase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; pY, phosphotyrosine.
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