From the ** Department of Immunology and
Department of Hematopoiesis, Holland Laboratory, American Red
Cross, Rockville, Maryland 20855,
Institute of
Biomedical Sciences, George Washington University Medical Center,
and ¶ Center for Biologics Evaluation and Research, Federal Drug
Administration, Bethesda, Maryland 20892
Received for publication, November 18, 2002
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ABSTRACT |
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Signal transducer and activator of transcription
6 (Stat6) plays an important role in interleukin (IL)-4-induced
responses. To analyze the regulation of Stat6 phosphorylation, cells
were cultured in the continuous presence of IL-4 or after a pulse and washout. In the continual presence of IL-4, Stat6 remained
phosphorylated for an extended period. After IL-4 removal and
inhibition of the Janus family kinase, tyrosine-phosphorylated Stat6
decayed at a rate dependent upon the length of IL-4 stimulation. The
decay of tyrosine-phosphorylated Stat6 was similar in the presence or absence of either cycloheximide or actinomycin D. In the absence of
functional Src homology-containing phosphatase-1 (SHP-1), the early
loss of tyrosine-phosphorylated Stat6 was substantially reduced.
Furthermore, the rate of loss of tyrosine-phosphorylated Stat6 in cells
expressing a mutation of the human IL-4 receptor Interleukin (IL)-41 is a
multifunctional cytokine that plays several critical roles in the
regulation of immune responses. IL-4 initiates these cellular responses
by binding to and signaling through a receptor complex that contains
the IL-4R Although it is recognized that the down-modulation of activated Stats
is important, few studies have focused on this aspect of the Jak/Stat
pathway. Tyrosine dephosphorylation of signaling proteins has been
recognized as one mechanism underlying the regulation of signal
transduction by many receptors. Recently, there has been progress in
delineating the role of phosphatases that bind to immunoreceptor
tyrosine-based inhibitory motif (ITIM) sequences in the cytoplasmic
domains of signaling receptors. ITIMs have been found in the
cytoplasmic domains of the Fc Several recent studies (15-18) indicate that Stat activation can be
regulated in a negative feedback loop. Just as the Jak/Stat signaling
pathways are activated by cytokines, certain negative regulatory
molecules also appear to be induced by cytokines. IL-4 can induce the
expression of several genes belonging to a family termed suppressors of
cytokine signaling (SOCS), including SOCS-1 in some cell types (17,
18). SOCS family members modulate signaling by several mechanisms that
include blocking access of the Stats to receptor-binding sites and
ubiquitination of the Janus kinases and their subsequent targeting to
the proteasome (19). Recently, it has been shown that the
de-ubiquitinating enzyme DUB-2 can prolong Stat5 phosphorylation
induced by IL-2 (20). The SOCS family is also believed to regulate the
Jak/Stat pathway by interfering with the function of the Jak kinases
(17, 18, 21). Furthermore, IL-2-induced SOCS-2 has been shown to inhibit IL-2 responses in a classical negative feedback loop (22). Overexpression of SOCS-1 in a murine B-cell line resulted in decreased Jak1 phosphorylation, decreased Stat6 phosphorylation, and decreased CD23 expression (15). SOCS proteins may be acting in a classic negative
feedback loop to suppress the Jak-mediated tyrosine phosphorylation of Stat6.
Although there is considerable understanding of the IL-4-mediated
tyrosine phosphorylation of Stat6, little is known about the regulation
of its inactivation. Therefore, to characterize the regulation of Stat6
tyrosine phosphorylation, we analyzed the rate of dephosphorylation
after IL-4 removal and Jak kinase inhibition in a variety of cell
types. The results presented herein indicate that the loss of
tyrosine-phosphorylated Stat6 is regulated by the rapid action of
a tyrosine phosphatase and the action of proteasome-mediated protein degradation.
Cell Culture--
Human epithelial lung carcinomas Calu-3 and
A549 were maintained in Dulbecco's modified Eagle's medium
(BioWhittaker, Inc., Walkersville, MD) supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Human monocytic THP-1 cells were maintained
in RPMI (BioWhittaker, Inc.) supplemented with 10% fetal bovine serum,
100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM
glutamine, and 0.05 mM 2-mercaptoethanol, 10 mM
HEPES, 4.5 g/liter glucose, and 1.0 mM sodium pyruvate. Human monocytic U937 cells were maintained in RPMI supplemented with
10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Murine B-cell lymphoma
M12.4.1 and murine T-hybridoma A1.1 were maintained in RPMI
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 0.05 mM 2-mercaptoethanol. IL-3-dependent myeloid
cell line 32D expressing the huIL-4R Reagents--
Recombinant murine IL-4 and recombinant huIL-4
were obtained from R & D Systems (Minneapolis, MN). AG490 was obtained
from Calbiochem and was stored at Lymphocyte Isolation--
Human blood was diluted in PBS, and
peripheral blood mononuclear cells were isolated by density gradient
centrifugation using Ficoll-Paque. Cells were activated with
plate-bound OKT3 (gift from Dr. Yufang Shi) for 72 h at 37 °C
to obtain primary activated T-lymphocytes. Human B-cells were purified
from peripheral blood mononuclear cells with the B-cell isolation kit
according to the manufacturer's protocol (Miltenyi Biotec, Auburn, CA).
Subcellular Fractionation--
Briefly, cells were stimulated
with IL-4 (10 ng/ml) at 37 °C and subjected to a pulse-washout
experiment. The cells were washed three times in complete 37 °C
media, resuspended at 2 × 106 cells/ml, and then
cultured in the absence of cytokine. Subcellular fractionation was
performed using NE-PER nuclear and Cytoplasmic extraction reagents
according to the manufacturer's protocol (Pierce).
Immunoprecipitation and Immunoblotting--
Briefly, cells were
stimulated with IL-4 (10 ng/ml) at 37 °C and subjected to a
pulse-washout experiment. The cells were washed three times in complete
37 °C media, resuspended at 2 × 106 cells/ml, and
then treated with sodium orthovanadate, MG132, lactacystin, actinomycin
D, or cycloheximide at 37 °C. The reaction was terminated by
dilution in ice-cold PBS, and cells were lysed in HEPES buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl,
0.5% Nonidet P-40, 50 mM NaF, 10 mM
pyrophosphate, 1 mM Na3VO4, and
protease inhibitors). The soluble fraction was immunoprecipitated with anti-Stat6 (Santa Cruz Biotechnology, Santa Cruz, CA). The precipitates were washed in lysis buffer and solubilized in SDS sample buffer. The
samples were separated on 7.5% SDS-polyacrylamide gels before transfer
to a polyvinylidene difluoride membrane. The membranes were then probed
with a monoclonal anti-phosphotyrosine antibody, RC20-HRPO
(Transduction Laboratories, Lexington, KY). The bound antibody was
detected using enhanced chemiluminescence (Kirkegaard & Perry
Laboratories, Gaithersburg, MD). Where indicated, the blots were
stripped and probed with control antibodies. Band intensities were
analyzed using the public domain software NIH Image. Data were graphed,
and slopes were analyzed using CA-Cricket Graph III.
RNA Isolation and Northern Blotting--
Total RNA was isolated
from M12.4.1 cells using RNA-Bee Isolation of RNA (Tel-Test, Inc.,
Friendswood, TX). Equivalent amounts of RNA (10 µg per lane)
were size-fractionated by electrophoresis in 1% agarose gels
containing 0.66 M formaldehyde. The RNA was then
transferred onto Nytran membranes and cross-linked by exposure to UV
light. The membranes were then hybridized and washed according to
standard procedures. The SOCS-1 probe was a 724-bp
XbaI fragment of a full-length murine SOCS-1 cDNA (16).
Gel-purified insert DNA was radiolabeled by the random-primer method.
Kinetics of Dephosphorylation of Stat6--
Even though Stat6 is
one of the major signaling molecules activated by IL-4, there has been
little research into the kinetics and regulation of Stat6 tyrosine
phosphorylation. It has been shown that B-cells must be in contact with
IL-4 for an extended period to become activated, and the minimum time
for half-maximal activation of biological responses is 12 h (27).
It has also been shown that transient Stat6 activation after exposure
to IL-4 is not sufficient to induce CD23 expression (28). In addition, B-cells produce little IgE unless IL-4 is present for at least 2 days
of culture (29). These responses require the activity of Stat6. It has
been reported that Stat6 DNA binding activity is rapidly activated in
B-cells by IL-4 and remains that way for up to 24 h (6). To
analyze the kinetics and longevity of Stat6 tyrosine phosphorylation,
we analyzed several different B-lymphocyte lines. M12.4.1 cells (a
murine B-lymphoma), Ramos cells (a human B-cell line), and normal human
B-cells purified from peripheral blood were treated with IL-4 for
various times, and tyrosine phosphorylation of Stat6 was analyzed by
immunoprecipitation and immunoblotting. In the continuous presence of
IL-4, Stat6 was rapidly phosphorylated and remained phosphorylated for
extended periods (up to 16 h in this experiment) (Fig.
1A). These results are
consistent with previous studies showing Stat6 activation in murine
B-cells for 24-48 h after IL-4 treatment (6) and suggest that if IL-4
is continuously present, the overall pool of Stat6 remains
tyrosine-phosphorylated.
As long as IL-4 is present, a constant level of tyrosine-phosphorylated
Stat6 is maintained. This implies the presence of an equilibrium
between the tyrosine phosphorylation of Stat6 and the dephosphorylation
of phospho-Stat6. The tyrosine phosphorylation of Stat6 will be
referred to as the forward reaction, and the dephosphorylation of Stat6
will be referred to as the reverse reaction of this cycle. Although it
is known that the Jaks drive the tyrosine phosphorylation of Stat6, it
is not known what mechanisms are involved in the dephosphorylation of
Stat6. To determine whether Stat6 could remain phosphorylated in the
absence of IL-4 signaling the forward reaction, we first determined the
concentration of AG490, an inhibitor of the Jak kinases, capable of
blocking the tyrosine phosphorylation of Stat6 (Fig. 1B). We
found that 25 µM AG490 blocked the tyrosine
phosphorylation of Stat6 by 83%, and 100 µM AG490
blocked tyrosine phosphorylation of Stat6 by 100%.
To determine whether Stat6 could remain phosphorylated in the absence
of IL-4 signaling, M12.4.1 cells were treated with IL-4 for either 15 min or 2 h; the cytokine was washed away, and the cells were then
cultured in the absence of cytokine but in the presence or absence of
50 µM AG490. The level of tyrosine-phosphorylated Stat6
in the cytoplasmic fraction was assessed at various time points
following cytokine removal and addition of AG490 (Fig. 2A). After removal of IL-4,
the levels of detectable Stat6 tyrosine phosphorylation declined over
time in a similar fashion in the presence or absence of AG490
indicating that the removal of IL-4 was sufficient to stop significant
forward phosphorylation. Similar results were obtained when an
anti-IL-4 antibody, 11B11, was used to terminate IL-4
signaling.3 The Western blots
were scanned; densitometry was performed using NIH-Image, and the ratio
of phosphorylated Stat6 to total Stat6 protein was calculated. This
ratio was plotted against time, and a t1/2 for
dephosphorylation was calculated (Fig. 2B). In M12.4.1
cells, the half-life of tyrosine-phosphorylated Stat6 after a 15-min
stimulation with IL-4 was 1.8 h (media slope =
In order to determine whether the above results are observed in more
than one cell type, several different IL-4-responsive cells of
hematopoietic and non-hematopoietic origins were analyzed in the same
manner as the M12.4.1 cells. A more rapid loss of tyrosine-phosphorylated Stat6 after a long stimulation than after a
short stimulation was also observed in U937, THP-1, and primary T-cells
(Table I). The length of IL-4 treatment
did not affect the length of tyrosine phosphorylation of Stat6 in A1.1,
A549, or Calu-3 cell lines (Table I). The relative levels of tyrosine phosphorylation at the zero time point for both the 15-min and 2-h
simulation periods were similar. These results suggest that as long as
IL-4 is present, Stat6 remains tyrosine-phosphorylated, and after IL-4
removal, the tyrosine phosphorylation of Stat6 declines over time. The
results also suggest that the half-life of tyrosine-phosphorylated
Stat6 can be dependent upon the length of IL-4 treatment in several
cell types.
It is possible that the regulation of tyrosine-phosphorylated Stat6
differs between cytoplasmic and nuclear compartments. Therefore,
M12.4.1 cells were stimulated with IL-4, and cytoplasmic and nuclear
fractions were prepared by subcellular fractionation. In the absence of
IL-4 signaling, Stat6 does not become tyrosine-phosphorylated, and
there is noticeably more total Stat6 protein in the cytoplasmic compartment compared with the nuclear compartment (Fig.
3A, 1st and
2nd lanes). Upon IL-4 signaling, Stat6 becomes
tyrosine-phosphorylated which is evident in both compartments (Fig.
3A, 3rd and 4th lanes), but there was
much more tyrosine-phosphorylated Stat6 in the cytoplasmic compartment
than the nuclear compartment. There was also an increase of total
nuclear Stat6 protein after IL-4 treatment over the amount observed in
the unstimulated nuclear fraction, which indicates some movement of
Stat6 from the cytoplasm into the nucleus upon IL-4 signaling.
To analyze the decay of tyrosine-phosphorylated Stat6 in the nuclear
compartment, M12.4.1 cells were treated with IL-4 for either 15 min or
2 h; the cytokine was washed away; the cells were then cultured in
the absence of cytokine, and subcellular fractionation was performed to
obtain the nuclear fraction of the cells. The level of
tyrosine-phosphorylated Stat6 was assessed at various time points
following cytokine removal and subcellular fractionation (Fig.
3B). As explained previously, the scans of the Western blots
were used to determine the ratio of tyrosine-phosphorylated Stat6 to
total Stat6 protein. The nuclear lysate was probed with Sp1, a nuclear
protein, to confirm proper subcellular fractionation. After removal of
IL-4, the level of detectable Stat6 tyrosine phosphorylation declined
over time in the nuclear compartment in a similar fashion to the
cytoplasmic compartment (compare Figs. 2 and 3). However, the half-life
of nuclear tyrosine-phosphorylated Stat6 after a 15-min or 2-h
stimulation with IL-4 was similar (t1/2 = 1.2).
These results suggest that the difference in the half-lives of
tyrosine-phosphorylated Stat6 between the 15-min
(t1/2 = 1.8) and 2-h (t1/2 = 1.2)
stimulation observed in Fig. 2 corresponds to a mechanism specific to
the cytoplasmic compartment.
The Loss of Tyrosine-phosphorylated Stat6 Is Not Dependent Upon New
Protein Synthesis--
The majority of investigations on cytokines has
focused on the mechanisms by which they activate signaling pathways.
However, it is clear that the actions of cytokines are limited in both magnitude and duration, making it important to understand the mechanisms by which their actions are negatively controlled (30). There
are several different mechanisms that could cause the decay of
IL-4-induced activated Stat6. The fact that a longer stimulation by
IL-4 resulted in a rapid loss of cytoplasmic tyrosine-phosphorylated Stat6 raised the possibility of a regulatory feedback loop. IL-4 stimulation could cause the expression of new genes that may act negatively upon IL-4 signaling, such as a SOCS family member. Previously, it has been shown that the IL-4-induced tyrosine
phosphorylation of Stat6 is not dependent on new protein synthesis (6).
In order to determine whether new protein synthesis is required for the
decay of tyrosine-phosphorylated Stat6, we stimulated cells with IL-4 for 15 min or 2 h in the presence or absence of
cycloheximide. Subsequently, IL-4 was removed, and the cells were
cultured in the absence of cytokine but in the presence or absence of
cycloheximide. As shown in Fig. 4, we
found that the decay of tyrosine-phosphorylated Stat6 was similar in
the presence or absence of cycloheximide. These results indicate that
induction of new protein synthesis is not required to regulate the
decay of tyrosine-phosphorylated Stat6. Furthermore, cycloheximide did
not alter the changes in the half-life of cytoplasmic
tyrosine-phosphorylated Stat6 induced by longer IL-4 treatment.
We also found that SOCS-1 mRNA was induced by IL-4 stimulation in
M12.4.1 cells and that this induction was blocked by actinomycin D
(Fig. 5A). Similar results
were obtained for U937 cells where the primary IL-4-induced gene was
cytokine-inducible SH2-containing protein.4 In order to
determine whether new mRNA synthesis is required for the decay of
tyrosine-phosphorylated Stat6, we stimulated cells with IL-4 for 2 h in the presence or absence of actinomycin D. Subsequently, IL-4 was
removed, and the cells were cultured in the absence of cytokine but in
the presence or absence of actinomycin D. As shown in Fig.
5B, we found the decay of tyrosine-phosphorylated Stat6 was
similar in the presence or absence of actinomycin D. These results
indicate that the induction of new mRNA is not required to regulate
the decay of tyrosine-phosphorylated Stat6. These data together with
the cycloheximide data suggest that SOCS proteins do not play a
significant role in the dephosphorylation of tyrosine-phosphorylated Stat6.
The Role of SHP-1 in the Decay of Tyrosine-phosphorylated
Stat6--
Recently there have been a number of studies on the role of
protein-tyrosine phosphatases in cytokine signaling in general, and
specifically in the IL-4-induced activation of Stat6. However, the
results have been conflicting, and the precise role for tyrosine phosphatases has not been clarified (13, 14, 31-33). Prolongation or
non-ligand-dependent activation of Stats can be observed in cells treated with pervanadate (34), indicating a role for
phosphotyrosine phosphatases in the activation-inactivation cycle. One
group suggested a potential role for cytoplasmic protein-tyrosine
phosphatases in directly dephosphorylating Stat proteins and in
maintaining a basal steady state level of Stat activity (12). In order
to determine whether tyrosine phosphatases are important in the decay rate of tyrosine-phosphorylated Stat6, we initially used sodium orthovanadate to inhibit tyrosine phosphatases. In the presence of
sodium orthovanadate, the loss of tyrosine-phosphorylated Stat6 at
early time points (
In order to determine whether SHP-1 can mediate the early loss of Stat6
tyrosine phosphorylation, Stat6 was analyzed in WT or SHP-1 mutant
(mev/mev)
IL-3-dependent hematopoietic precursor cell lines. WT and
mev/mev cells were stimulated
with IL-4 for 2 h; the cytokine was removed; the cells were
cultured in the absence of cytokine, and then subcellular fractionation
was performed to obtain cytoplasmic and nuclear compartments. The loss
of tyrosine-phosphorylated Stat6 in SHP-1 mev/mev cells was greatly delayed
compared with WT cells at early time points (t
These data suggest a role for protein-tyrosine phosphatases in the
regulation of Stat6 activation and specifically SHP-1 in cells of
hematopoietic origin. We showed previously (24) that transfected
huIL-4R
It has been suggested that SHP-1 may negatively regulate the Jak family
tyrosine kinases (35). It is possible that the results presented so far
could be accounted for by continual Jak kinase activity and concomitant
new Stat6 phosphorylation. Therefore, to verify that we are analyzing
only the decay of tyrosine-phosphorylated Stat6 and not residual Jak
activity, SHP-1 mev/mev cells and
32D Y5F cells were simulated with murine or human IL-4 as appropriate
for 2 h; the cytokine was removed, and the cells were cultured in
the absence of cytokine but in the presence or absence of 50 µM AG490. The level of tyrosine-phosphorylated Stat6 was
assessed at various time points following cytokine removal. The rate of
loss of tyrosine-phosphorylated Stat6 in each of the mutant cell lines
was equal in the presence or absence of AG490 (Fig.
8). Similarly, AG490 had no effect on the
loss of tyrosine-phosphorylated Stat6 in the WT cell
lines.3 The differences observed in Figs. 6 and 7 between
WT and mutant cells are not simply due to residual Jak activity.
MG132 Enhances the Longevity of Tyrosine-phosphorylated
Stat6--
Another mechanism that could cause the decay of
tyrosine-phosphorylated Stat6 is the degradation of proteins involved
in the Jak/Stat pathway in the cytoplasm or nucleus. Inhibitors of
proteasome activity have been shown to prolong the activation of Stat1
in response to interferon- The IL-4-induced activation of Stat6 is clearly involved in many
biologic responses related to allergy. Recent evidence has emphasized
the importance of regulatory pathways that function to modulate
intracellular signals initiated by IL-4 and other cytokines. In the
last few years, the majority of research on the actions of cytokines
has focused on the mechanisms by which they initiate signaling
pathways. However, it is known that the actions of cytokines can be
limited in both magnitude and duration. This could be especially
important in the control of cytokine action in vivo. Even
though it is apparent that tyrosine-phosphorylated Stat6 plays an
important role in many IL-4-induced responses; there has not been much
research on determining the mechanisms that regulate the longevity of
Stat6 activation. Therefore, a clear understanding of the
down-regulation of tyrosine-phosphorylated Stat6 is essential.
In the continuous presence of IL-4, Stat6 is rapidly phosphorylated and
remains that way for an extended period. However, after the IL-4
stimulus is removed, the levels of tyrosine-phosphorylated Stat6
decline over time. These results indicate that when IL-4 is present to
signal there is equilibrium between the Jak-dependent forward reaction of tyrosine phosphorylation and the reverse reaction of tyrosine dephosphorylation (Fig.
10). By including AG490 in our
experiments, we would inhibit any residual forward activity and
specifically focus on the possible mechanisms of the tyrosine dephosphorylation of Stat6 (the reverse reaction).
in the
immunoreceptor tyrosine-based inhibitory motif sequence (Y5F) was
dramatically decreased compared with wild-type cells. The early rate of
decay was similar in the presence or absence of MG132, an inhibitor of
the proteasome, but the later rate of decay was decreased 5-fold. These
results suggest that the loss of tyrosine phosphorylation of Stat6 is
regulated by the action of SHP-1 and the proteasome but is not
dependent on new protein synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain and the
common chain (1). This results in a
series of phosphorylation events that occur through the activation of
the Janus kinase/signal transducer and activator of transcription
(Jak/Stat) pathway (1-3). IL-4 engagement of the receptor leads to
tyrosine phosphorylation of Jak-1 and Jak-3, which leads to the
tyrosine phosphorylation of the IL-4R
chain itself. The
phosphorylated tyrosine residues act as docking sites for signaling
molecules such as Stat6. Stat6 is a latent cytoplasmic transcription
factor necessary for the induction of expression of IL-4-responsive
genes (reviewed in Ref. 1) and is the primary Stat activated in
response to IL-4 stimulation (2-4). It has been shown clearly that the
functional activity of Stat6 is dependent on tyrosine phosphorylation,
and numerous studies (1, 4-6) have analyzed the events leading to its activation.
RIIb1 immunoglobulin receptor, killer
inhibitory receptors, and cytotoxic T-lymphocyte antigen-4 molecules
and function in the generation of negative regulatory signals by these
receptors (7-11). There is also recent data linking tyrosine
phosphatases and Stats. One group (12) has demonstrated that the
SH2-containing phosphatase-2 (SHP-2), but not SHP-1, directly
dephosphorylates cytoplasmic Stat5 in an in vitro tyrosine
phosphatase assay with purified proteins. There have been several
studies analyzing the role of SHP-1 in Stat6 activation; however, the
reports contradict one another. One report (13) states that Stat6
activation by IL-4 is significantly enhanced in pre-B-cells derived
from motheaten (SHP-1 mutant) mice compared with normal
pre-B-cells derived from control animals. Alternatively, another report
(14) states that SHP-1 is not a negative regulator of IL-4 signaling in
bone marrow-derived mast cells. Although these overall results differ,
they suggest that there may be a role for protein-tyrosine phosphatases
in regulating Jak/Stat pathways.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
wild-type (WT) or a Y713F
mutation (23, 24) were maintained in RPMI supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and
5% WEHI-3B supernatant. Murine SHP-1 WT and mutant cells are
IL-3-dependent primitive hematopoietic precursor cells
isolated from the yolk sac (9.0-9.5 days post-coitum) of WT or
mev/mev embryos and immortalized
by a homeobox-containing gene Hox11 (25,
26).2 They were maintained in
RPMI supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 0.05 mM 2-mercaptoethanol, and 10% WEHI-3B supernatant.
20 °C in
Me2SO. MG132 was obtained from Biomol (Plymouth
Meeting, PA) and was stored at
80 °C in Me2SO.
Cycloheximide was obtained from Sigma and was dissolved in PBS prior to
use. Actinomycin D was obtained from Biomol and was stored at
20 °C in ethanol. Lactacystin, clasto-lactacystin
-lactone, Z-LLF-CHO, Z-Val-Phe-CHO, and ALLM were all obtained from
Calbiochem and were dissolved in Me2SO prior to use. Sodium orthovanadate was obtained from ICN (Aurora, OH) and was dissolved in
PBS prior to use. Anti-IL-4 monoclonal antibody, 11B11, was a gift from
C. Watson and Dr. W. E. Paul (Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stat6 phosphorylation in B-cells.
A, M12.4.1 cells, Ramos cells, and primary human B-cells
were treated with 10 ng/ml IL-4 (murine (m) or human) for
the indicated times. Stat6 was immunoprecipitated (IP) from
whole cell lysates using anti-Stat6 antibody. Immunoprecipitates were
analyzed by Western blot (IB) using the anti-phosphotyrosine
antibody RC20-HRPO. The blot was stripped and reprobed using an
anti-Stat6 antibody. B, M12.4.1 cells were stimulated with
10 ng/ml murine IL-4 in the presence or absence of various
concentrations of AG490. Stat6 was immunoprecipitated from whole cell
lysates using anti-Stat6 antibody. Immunoprecipitates were analyzed by
Western blot using the anti-phosphotyrosine antibody RC20-HRPO. The
blot was stripped and reprobed using an anti-Stat6 antibody.
0.37, AG490
slope =
0.40), compared with a half-life of 1.2 h after a
2-h IL-4 stimulation (media slope =
0.38, AG490 slope =
0.36).
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Fig. 2.
Decay of Stat6 phosphorylation in B-cells
after IL-4 removal. A, M12.4.1 cells were treated with
10 ng/ml murine IL-4 for either 15 min or 2 h; the cytokine was
washed away, and the cells were cultured in the absence of cytokine but
in the presence or absence of 50 µM AG490 for the
indicated times. Stat6 was immunoprecipitated from whole cell lysates
using anti-Stat6 antibody. Immunoprecipitates were analyzed by Western
blot using the anti-phosphotyrosine antibody RC20-HRPO. The blot was
stripped and reprobed using an anti-Stat6 antibody. B,
densitometry was performed using NIH Image 1.62 in order to calculate
the ratio of phosphorylated Stat6 protein to total Stat6 protein.
CA-Cricket Graph III was used to generate the best-fit lines. The slope
of the line reflects the rate at which detectable tyrosine
phosphorylation of Stat6 is lost. Time to achieve a 50% loss of
tyrosine-phosphorylated Stat6 was calculated at a
t1/2. Results are representative of three
experiments.
Decay of Stat6 phosphorylation in various cell types
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Fig. 3.
Nuclear decay of Stat6 phosphorylation in
B-cells after IL-4 removal. A, M12.4.1 cells were
treated with 10 ng/ml murine IL-4, and subcellular fractionation was
performed using the Pierce NE-PER nuclear and cytoplasmic extraction
reagents according to the manufacturer's protocol to obtain
cytoplasmic (C) and nuclear (N) fractions. Stat6
was immunoprecipitated from the fractionated cell lysates using
anti-Stat6 antibody. Immunoprecipitates were analyzed by Western blot
using the anti-phosphotyrosine antibody RC20-HRPO. The blot was
stripped and reprobed using an anti-Stat6 antibody. B,
M12.4.1 cells were treated with 10 ng/ml murine IL-4 for either 15 min
or 2 h; the cytokine was washed away; the cells were cultured in
the absence of cytokine for the indicated times, and subcellular
fractionation was performed using the Pierce NE-PER nuclear and
cytoplasmic extraction reagents according to the manufacturer's
protocol. The nuclear lysate was probed with Sp1, a nuclear protein. As
in Fig. 2, the scans of the Western blots were used to determine the
ratio of tyrosine-phosphorylated Stat6 to total Stat6 protein.
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Fig. 4.
Decay of Stat6 phosphorylation does not
depend on new protein synthesis. M12.4.1 cells were treated with
10 ng/ml murine IL-4 for either 15 min or 2 h in the presence or
absence of cycloheximide (0, 10, or 20 µg/ml). The cytokine was
washed away, and the cells were cultured in the absence of cytokine but
in the presence or absence of cycloheximide for the indicated times. As
in Fig. 2, the scans of the Western blots were used to determine the
ratio of tyrosine-phosphorylated Stat6 to total Stat6 protein. Results
are representative of three experiments.
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Fig. 5.
SOCS-1 mRNA induced by IL-4 but blocked
by actinomycin D. A, M12.4.1 cells were treated with 10 ng/ml murine IL-4 in the presence or absence of 10 µg/ml actinomycin
D (Act.D) for 3 h. Total RNA was isolated and analyzed
by Northern blotting for SOCS-1. The blots were stripped and reprobed
for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
B, M12.4.1 cells were treated with 10 ng/ml murine IL-4 in
the presence or absence of actinomycin D (0, 5, or 10 µg/ml) for
2 h. The cytokine was washed away, and the cells were cultured in
the absence of cytokine but in the presence or absence of actinomycin
for the indicated times. As in Fig. 2, the scans of the Western blots
were used to determine the ratio of tyrosine-phosphorylated Stat6 to
total Stat6 protein. Results are representative of three
experiments.
1 h) was reduced,3 suggesting a role
for protein-tyrosine phosphatases in the early phase of
tyrosine-phosphorylated Stat6 decay.
1 h) in both the cytoplasmic and nuclear compartments (Fig.
6). In the absence of functional SHP-1
phosphatase, the early loss (t
1 h) of
tyrosine-phosphorylated Stat6 was almost eliminated. However, at later
time points (t
1 h), the levels of tyrosine-phosphorylated
Stat6 began to decline. These results indicate that SHP-1 does play a
role in the early (t
1 h) loss of tyrosine-phosphorylated
Stat6 in these cells.
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Fig. 6.
SHP-1 is important for the early loss of
tyrosine-phosphorylated Stat6 in hematopoietic precursor cells.
SHP-1 wild-type (SHP-1 WT) and mutant
mev/mev (SHP-1
MT) cells were treated with 10 ng/ml murine IL-4 for 2 h; the
cytokine was washed away; the cells were cultured in the absence of
IL-4 for the times indicated, and subcellular fractionation was
performed using the Pierce NE-PER nuclear and cytoplasmic extraction
reagents according to the manufacturer's protocol. Cytoplasmic and
nuclear lysates were immunoprecipitated and immunoblotted. As in Fig.
2, the scans of the Western blots were used to determine the ratio of
tyrosine-phosphorylated Stat6 to total Stat6 protein.
with a mutation of the ITIM sequence (Y5F) was able to
signal the tyrosine phosphorylation of Stat6 to the same degree as WT
huIL-4R
in IL-3-dependent 32D cells when human IL-4 was
continuously present. To test the contribution of this tyrosine residue
to the loss of tyrosine-phosphorylated Stat6, we stimulated 32D WT and
Y5F cells with human IL-4 for 2 h; the cytokine was removed; the
cells were cultured in the absence of cytokine, and then subcellular
fractionation was performed to obtain the cytoplasmic and nuclear
compartments. Strikingly, the rate of loss of tyrosine-phosphorylated
Stat6 in the Y5F mutant cells was dramatically decreased compared with
that of the WT cells in both the cytoplasmic and nuclear compartments
(Fig. 7). These results indicate that the
ITIM-tyrosine, Tyr-5, in the IL-4R
is important in regulating
the loss of tyrosine-phosphorylated Stat6.
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Fig. 7.
Mutation of tyrosine 5 in the human
IL-4R chain decreases the rate of decay of
tyrosine-phosphorylated Stat6 in 32D cells. 32D cells expressing
WT huIL-4R
and 32D cells expressing huIL-4R
with a mutation in
tyrosine 5 (Y5F) were treated with 10 ng/ml human IL-4 for 2 h;
the cytokine was washed away; the cells were cultured in the absence of
IL-4 for the times indicated, and subcellular fractionation was
performed using the Pierce NE-PER nuclear and cytoplasmic extraction
reagents according to the manufacturer's protocol. Cytoplasmic and
nuclear lysates were immunoprecipitated and immunoblotted. As in Fig.
2, the scans of the Western blots were used to determine the ratio of
tyrosine-phosphorylated Stat6 to total Stat6 protein.
View larger version (37K):
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Fig. 8.
Presence of AG490 does not affect the decay
of tyrosine-phosphorylated Stat6 in SHP-1
mev/mev cells or 32D
Y5F cells. SHP-1
mev/mev cells (A)
or 32D Y5F (B) cells were simulated with 10 ng/ml murine or
human IL-4 as appropriate for 2 h; the cytokine was washed away,
and the cells were cultured in the absence of cytokine but in the
presence or absence of 50 µM AG490 for the times
indicated. Total lysates were immunoprecipitated and immunoblotted. As
in Fig. 2, the scans of the Western blots were used to determine the
ratio of tyrosine-phosphorylated Stat6 to total Stat6 protein.
(36, 37). One group found that in the
continuous presence of cytokine, the down-regulation of phosphorylated Stat4, Stat5, and Stat6 was inhibitable by the proteasome inhibitors MG132 and lactacystin (38). To assess the contribution of the proteasome on the dephosphorylation of Stat6, M12.4.1 cells were treated with IL-4 for 2 h; the cytokine was removed, and the cells were cultured in the absence of cytokine but in the presence or absence
of MG132 (Fig. 9A). The early
rate (t
3 h) of decay was similar in the presence
(slope =
0.21) or absence (slope =
0.20) of MG132.
However, after 3 h, the levels of tyrosine-phosphorylated Stat6
were stabilized by MG132 (the slope of the line shifted from
0.21 to
a slope of
0.04) (Fig. 9B). There was no effect of this
inhibitor on the levels of total Stat6 protein. Similar results were
obtained using other proteasome inhibitors including lactacystin (a
specific proteasome inhibitor that does not inhibit any other
proteases), clasto-lactacystin
-lactone (a more potent form of lactacystin), or Z-LLF-CHO (another potent and cell-permeable proteasome inhibitor) but not with specific inhibitors of calpain or
cathepsins B or L such as Z-Val-Phe-CHO (inhibits calpain I and II) or
ALLM (inhibits calpain I and II and cathepsin B and L).3
These results confirm that tyrosine-phosphorylated Stat6 is partially regulated by the action of proteasome-mediated protein degradation.
View larger version (29K):
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Fig. 9.
MG132 decreases the late rate of decay of
tyrosine-phosphorylated Stat6. A, M12.4.1 cells were
treated with 10 ng/ml murine IL-4 for 2 h; the cytokine was washed
away, and the cells were cultured in the absence of IL-4 but in the
presence or absence of 30 µM MG132 for the times
indicated. Total lysates were immunoprecipitated and immunoblotted.
B, as in Fig. 2, the scans of the Western blots were used to
determine the ratio of tyrosine-phosphorylated Stat6 to total Stat6
protein. Results are representative of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Cycle of tyrosine phosphorylation.
Major players of Stat6 tyrosine phosphorylation and dephosphorylation
are indicated.
A 2-h stimulation with IL-4 resulted in a surprisingly rapid loss of tyrosine-phosphorylated Stat6 in some cell types. This observation raised the question of whether IL-4 stimulation was causing the synthesis of regulatory proteins that could in turn act negatively upon IL-4 signaling. A natural candidate for a regulatory protein acting upon IL-4 signaling would be a member of the SOCS family. However, it was found that SOCS-1 does not play a major role in the turnover of tyrosine-phosphorylated Stat6 in M12.4.1 cells. At first glance, this may seem to contradict previous findings on the involvement of SOCS proteins in IL-4 signaling (15, 16, 39). Research thus far indicates that SOCS proteins are important for IL-4 signaling in the forward reaction of the Stat6 activation cycle and likely act to suppress directly Jak activity (Fig. 10). For example, it has been demonstrated that interferons inhibit IL-4-induced activation of Stat6 and Stat6-dependent gene expression at least in part by inducing expression of SOCS-1 (16). However, our results suggest that SOCS-1 does not play a significant role in Stat6 tyrosine dephosphorylation, the reverse reaction.
Several studies have indicated that tyrosine phosphatases play a role
in regulating Stats. Whereas the IL-4R does contain an ITIM
consensus sequence (a conserved Tyr-713 surrounded by a proline-rich
sequence) (23) shown to interact with SHP-1 in vitro, its
role in regulating Stat6 phosphorylation remains unclear (33). One
report stated that Stat6 activation by IL-4 is significantly enhanced
in pre-B-cells derived from motheaten (SHP-1 mutant) mice,
compared with normal pre-B-cells derived from control animals (13).
However, hematopoiesis is greatly altered in the motheaten mice, which affects the cellular phenotype and activation state of the
isolated cell populations (40). Therefore, it is not known whether the
differences seen between the SHP-1 mutant mice and the wild-type mice
are due to differences in SHP-1 activity or defects in the cells
themselves. Another report (14) stated that SHP-1 is not a negative
regulator of IL-4 signaling in bone marrow-derived mast cells. Their
research revealed that mast cells derived from motheaten
mice do not exhibit enhanced or prolonged Stat6 activation in the
continuous presence of IL-4 when compared with wild-type cells. We
found that sodium orthovanadate had an early effect in stabilizing the
tyrosine-phosphorylated form of Stat6. Furthermore, in the absence of
functional SHP-1, tyrosine-phosphorylated Stat6 was quite stable.
Motheaten (me/me) and viable motheaten (mev/mev) mice contain a
mutation in the coding region for the N-terminal SH2 domain and the
catalytic domain of SHP-1, respectively (41, 42). As a result of these
mutations, me/me mice die of multiple complex
hematological and immunological diseases. We used WT and mev/mev primitive
hematopoietic precursor cells isolated from early yolk sac to avoid the
potential secondary deteriorating changes with the hematopoietic cells
isolated from adult motheaten mice that display multiple
hematological abnormalities. Cells were also treated with AG490 to
block the Jak kinases and to ensure that we were analyzing the decay of
tyrosine-phosphorylated Stat6. These studies indicated that SHP-1 plays
a role in the reverse reaction of Stat6 tyrosine phosphorylation in
hematopoietic cells. Interestingly, we found that SHP-1 mutant
mev/mev cells were also more
sensitive to low concentrations of IL-4 during the forward reaction of
the cycle of tyrosine phosphorylation of Stat6.3 These
results suggest that SHP-1 may also play a role in the forward reaction
in the cycle of Stat6 tyrosine phosphorylation.
To confirm SHP-1 as a player in the cycle of tyrosine phosphorylation
of Stat6, cells expressing the huIL-4R with a mutated 5th tyrosine
(ITIM site) were analyzed. It was possible that the mutation of this
tyrosine would result in the loss of recruitment of tyrosine
phosphatases, such as SHP-1, to the receptor because the site would no
longer become phosphorylated upon ligand binding. Strikingly, the rate
of loss of tyrosine phosphorylation of Stat6 in the Y5F mutant cells
was dramatically decreased compared with that of the WT cells.
Therefore, the 5th tyrosine residue in the IL-4R
chain is important
for regulation of Stat6. Our previous results (24) suggest that the 5th
tyrosine does not play a role in the forward reaction. However, by
specifically analyzing the dephosphorylation phase by treating cells
with AG490, we revealed that this residue plays a dramatic role in the
reverse reaction.
There have been many studies on the IL-4-induced activation of Stat6; however, each study has usually been performed with a single cell type (5, 6, 23, 24). The activation of Stat6 in monocytic THP-1 cells was examined after IL-4 treatment, and it was found that the activation was rapid and transient (5). Another group (13) noted that IL-4-dependent activation of Stat6 is sustained longer in fibroblast cells than in lymphoid cells. These reports suggest that tyrosine-phosphorylated Stat6 may have a short half-life in some cells types, although a direct comparison of different cell types has not been performed before. Potential differences may have direct relevance to the potency of in vivo effects of IL-4. The cells of hematopoietic lineage all seem to follow a similar trend, a long stimulation resulting in a rapid loss of tyrosine-phosphorylated Stat6 in the cytoplasm, although this is not evident in the nucleus. SHP-1, shown herein to be important in the loss of tyrosine-phosphorylated Stat6, is predominantly expressed in cells of hematopoietic lineage (41). However, all cell types did show a loss of tyrosine-phosphorylated Stat6 after removal of cytokine. Therefore, there must be other tyrosine phosphatases playing a role in the regulation of Stat6 in non-hematopoietic cells.
In contrast to the phosphatase inhibitors, the proteasome inhibitors
had effects at later time points (t 3 h). MG132,
lactacystin, and other inhibitors stabilized the
tyrosine-phosphorylated form of Stat6 after an initial loss. It should
be noted that there was no loss of total Stat6 protein in
these experiments, so it is likely that the proteasome is not acting on
Stat6 itself. It may be acting on other as yet uncharacterized proteins
involved in the dephosphorylation phase. The data herein present the
possibility that two independent phases contribute to the loss of
tyrosine-phosphorylated Stat6. The protein-tyrosine phosphatases may
play early roles (t
1 h) in the loss of
tyrosine-phosphorylated Stat6, and the proteasome may play a more
important role in the later loss (t
3 h) of
tyrosine-phosphorylated Stat6. Therefore, proteasome-mediated protein
degradation is another possible mechanism that could play a role in the
reverse reaction of the cycle of tyrosine phosphorylation of Stat6.
Recent studies (28) have suggested that the amount of cytosolic tyrosine-phosphorylated Stat6 may influence the amount of Stat6 able to translocate into the nucleus to activate gene expression. Therefore, the regulation of the amount of cytosolic tyrosine-phosphorylated Stat6 would be important. If tyrosine-phosphorylated Stat6 is depleted from the cytoplasmic pool by phosphatases or by proteasome-mediated protein degradation, then the nuclear pool of tyrosine-phosphorylated Stat6 would be affected. It has been proposed that tyrosine-phosphorylated Stat6 is imported into the nucleus at the same rate it is exported (28). Therefore, a depletion of tyrosine-phosphorylated Stat6 in the cytoplasm would mean that little or no tyrosine-phosphorylated Stat6 could be imported into the nucleus. At the same time, the Stat6 in the nucleus would still be exported out to the cytoplasm at the same rate such that a depletion in the cytoplasm would eventually lead to a depletion in the nucleus. In addition, the action of nuclear tyrosine phosphatases, including SHP-1, would enhance the loss of nuclear tyrosine-phosphorylated Stat6 (43-46).
The importance of these Stat6-regulating mechanisms on the magnitude of
in vivo responses is not yet clear. The production of
cytokines is not similar to production of endocrine hormones, but
rather more similar to the production of neurotransmitters (47). For
example, when helper T-cells interact with B-cells, IL-4 is secreted in
a directional manner in a small, confined space created by close
apposition of the B- and T-cell membranes, the so-called immunological
synapse (47, 48). This cellular interaction would in theory be of
relatively short duration in vivo (49), perhaps making the
exposure of target cells to IL-4 more akin to a pulse than to a
continuous exposure. This would also be true for IL-4 released from
mast cells or basophils in response to FcRI cross-linkage. If,
indeed, the in vivo situation is analogous to an in
vitro IL-4 pulse, then any mutations or alterations in the
dephosphorylation of Stat6 should have a potent effect on IL-4-mediated
responses. Of special interest are the polymorphisms of the human
IL-4R
in the cytoplasmic domain that have been associated with
allergy and asthma (33, 50). Analyses of the ability of these
polymorphic receptors to signal in various transfection systems have
not yet revealed any major changes in signaling efficiency, although
the previous studies (24) were done in the continuous presence of IL-4.
In this study, we found that differences between the Y5F mutant and WT
were not revealed until we utilized an IL-4 pulse treatment protocol.
Therefore, it will be important to analyze specifically the impact of
IL-4R
polymorphisms on regulation of Stat6 dephosphorylation in
normal cell types.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Elena Semenova, Xuilan Qi, and Dr. Ann Welch for technical support in the laboratory, Dr. Ricki Helm for discussion and critical reading of the manuscript, and Dr. Mark Williams for technical advice and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI38985 (to A. D. K.), by The George Washington Institute of Biomedical Sciences (to E. M. H.), and by the American Red Cross.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.
§ Submitted this work in partial fulfillment of the requirements for the Doctor of Philosophy degree, Immunology Program, Institute for Biomedical Sciences of the Columbian School of Arts and Sciences, The George Washington University.
To whom correspondence should be addressed: Dept. of
Immunology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-517-0326; Fax: 301-517-0344; E-mail: KeeganA@usa.redcross.org.
Published, JBC Papers in Press, November 28, 2002, DOI 10.1074/jbc.M211747200
2 W.-M. Yu, T. S. Hawley, R. G. Hawley, and C.-K. Qu, submitted for publication.
3 E. M. Hanson, unpublished observations.
4 H. Dickensheets and R. P. Donnelly, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
Jak, Janus family kinase;
Stat, signal transducer and activator of
transcription;
ITIM, immunoreceptor tyrosine-based inhibitory motif;
SH2, Src homology;
SHP, SH2-containing phosphatase;
SOCS, suppressor of
cytokine signaling;
WT, wild-type;
me/me, motheaten;
mev/mev, viable
motheaten;
huIL-4R, human IL-4 receptor
;
Y5F, ITIM tyrosine
number 5 mutation to phenylalanine;
Fc
RI, Fc
receptor 1;
Z, benzyloxycarbonyl;
PBS, phosphate-buffered saline.
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