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Molecular and Cellular Biology, May 1999, p. 3727-3735, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Distinct Mechanisms of Activation of Stat1 and Stat3 by
Platelet-Derived Growth Factor Receptor in a Cell-Free
System
Marie-Luce
Vignais1,2,
and
Michael
Gilman1,3,*
Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 117241; Institut de
Génétique Moléculaire, Montpellier,
France2; and ARIAD Pharmaceuticals Inc.,
Cambridge, Massachusetts 021393
Received 30 December 1998/Accepted 22 February 1999
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ABSTRACT |
Ligand-dependent activation of the platelet-derived growth factor
receptor (PDGFR) in fibroblasts in culture leads to the activation of
the JAK family of protein-tyrosine kinases and of the transcription
factors Stat1 and Stat3. To determine the biochemical mechanism of STAT
activation by PDGFR, we devised a cell-free system composed of a
membrane fraction from cells overexpressing PDGFR. When supplemented
with crude cytosol, the membrane fraction supported PDGF- and
ATP-dependent activation of both Stat1 and Stat3. However, the extent
of Stat3 activation differed depending on the source of the cytosolic
fraction. Using purified recombinant STAT proteins produced in
Escherichia coli, we found that Stat1 could be activated by
immunopurified PDGFR and showed no additional requirement for membrane-
or cytosol-derived proteins. In contrast, activation of Stat3 exhibited
a strong requirement for the cytosolic fraction. The activity present
in the cytosolic fraction could be depleted with antibodies to JAK
proteins. We conclude that the mechanisms of activation of Stat1 and
Stat3 by PDGFR are distinct. Stat1 activation appears to result from a
direct interaction with the receptor, whereas Stat3 activation
additionally requires JAK proteins.
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INTRODUCTION |
Proteins of the STAT family of
transcription factors are activated by a wealth of cytokines and
polypeptide growth factors. Activation by cytokine receptors requires
members of the JAK family of protein tyrosine kinases (2-4,
8-10, 15, 17, 24). The general model for STAT activation was
based on the paradigm of STAT activation by the interferon receptors
(5, 18). Gamma interferon activates both Jak1 and Jak2,
which are in turn required for the activation of Stat1 (12,
25). Alpha interferon activates Jak1 and Tyk2, leading to the
activation of Stat1 and Stat2 (12, 21). For interferons and
other cytokines that act through cell-surface receptors that lack
intrinsic protein-tyrosine kinase activity, receptor-associated JAKs
provide the catalytic activity responsible for receptor phosphorylation
to create docking sites for signaling proteins such as STATs and
presumably for phosphorylation of the proteins recruited to the
phosphorylated receptor.
Polypeptide growth factors, such as epidermal growth factor and
platelet-derived growth factor (PDGF), also activate JAKs and
STATs (11, 22). Because the receptors for these factors harbor intrinsic protein-tyrosine kinase activity, the role of receptor-activated JAKs, if any, in STAT activation by these
receptors remains unclear. We used a cell-free system and
partially purified proteins to examine the biochemical steps in STAT
activation by the PDGF receptor (PDGFR). We found that the
mechanisms of activation of Stat1 and Stat3 are different and that JAKs
may be required only for the activation of Stat3.
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MATERIALS AND METHODS |
Cell cultures and cellular extracts.
The cell line 2fTGH.PS1
(22), derived from the human fibrosarcoma HT1080 cells, was
grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS).
Cytosolic extracts were prepared from actively growing subconfluent
cells starved for 15 to 18 h in DMEM supplemented with 0.5% FBS.
Cells were scraped from the dishes into phosphate-buffered saline
containing 2 mM Na3VO4 and resuspended in
hypotonic buffer (20 mM HEPES [pH 7.9]; 20 mM NaF; 1 mM
Na2P2O7; 1 mM EDTA; 1 mM EGTA; 0.25 mM ammonium molybdate; 2 mM Na3VO4; 3-µg/ml
concentrations of leupeptin, aprotinin, and pepstatin; 0.5 mM
phenylmethylsulfonyl fluoride [PMSF]; and 1 mM dithiothreitol
[DTT]. Cells were lysed by repeated passage through a 25-gauge
needle, and cytosolic extracts were prepared as described previously
(22).
Membrane fractions were prepared from confluent 2fTGH.PS1 dishes
starved for 24 h in DMEM supplemented with 0.5% FBS. Cells
were
scraped from the dishes into phosphate-buffered saline containing
2 mM
Na
3VO
4, resuspended in hypotonic buffer, and
lysed by repeated
passage through a 25-gauge needle. The extract was
spun twice
for 1 min to eliminate crude nuclei and unbroken cells and
supplemented
with NaCl to 120 mM. Membranes were sedimented by
centrifugation
(20 min at 16,000 ×
g), and the
membrane pellet was collected
after two additional washes in hypotonic
buffer. The 2fTGH.PS1
membrane pellets were resuspended in MRB buffer
(20 mM Tris [pH
7.4]; 20 mM NaF; 1 mM EDTA; 150 mM NaCl; 10%
glycerol; 2 mM Na
3VO
4;
1-µg/ml concentrations
of leupeptin, aprotinin, and pepstatin;
0.5 mM PMSF; 1 mM DTT; and
0.25%
Triton).
In vitro kinase assay.
The membrane fraction (20 µg/3
µl) was mixed with the STAT proteins and PDGF (PDGF-BB; Upstate
Biotechnology Inc.) (6 ng). STAT proteins were either of cytosolic
origin (12.5 to 25 µg of cytosolic extract) or purified from
Escherichia coli (about 20 ng of the pure protein). The
kinase reaction was initiated by the addition of a 5× kinase buffer
(50 mM HEPES [pH 7.9], 50 mM MgCl2, 10 mM ATP). The
reaction mixture (12.5 µl) was incubated at 30°C for 30 min and
stopped by the addition of EDTA (15 mM final concentration). The
reactions were spun for 5 min, and the supernatants (12 µl) were used
for the electrophoretic mobility shift assay (EMSA).
EMSA.
Protein fractions activated in vitro were preincubated
for 15 min at room temperature with 1 µg of poly(dI-dC) · poly(dI-dC) as nonspecific DNA competitor in the reaction buffer (13 mM
HEPES [pH 7.9], 65 mM NaCl, 1 mM DTT, 0.15 mM EDTA, 2% Ficoll 400). The labeled probe (~3 fmol) was then added to the reaction, which was
incubated for another 20 min at room temperature. The protein-DNA complexes were separated by electrophoresis in 0.5× TBE
(Tris-borate-EDTA) buffer through a 5% polyacrylamide gel
(acrylamide-to-bis ratio, 39:1) containing 2.5% glycerol. Gels were
dried and analyzed by autoradiography. The supershift experiment was
performed using monoclonal antibody 12CA5 (7).
Immunoprecipitations and Western blots.
For immunoblot
analysis of 2fTGH.PS1 or HeLa cytosolic extracts, proteins were
denatured in Laemmli buffer, separated by sodium dodecyl sulfate-7.5%
polyacrylamide gel electrophoresis and transferred to nitrocellulose.
Western blots were performed with monoclonal antibodies against Stat1
or Stat3 N-terminal regions (Transduction Laboratories), phospho-Stat1
(Y701) (New England Biolabs, Inc.), or hemagglutinin (12CA5)
(7). Immunoreactive bands were visualized with the
epichemiluminescence Western blotting system (Amersham).
For the depletion of U3A (
13) cytosol, protein extracts were
incubated for 3 h at 4°C with polyclonal antibodies (Upstate
Biotechnology Inc.) against JAK1, JAK2, Tyk2, or ERK2 prior to
the
addition of protein A Sepharose beads. For the immunoprecipitation
of
the PDGF-
receptor, membrane pellets from 2fTGH.PS1 cells
were
solubilized in MRB buffer containing 1% Triton and incubated
with
polyclonal antibodies against the human PDGF type B receptor
(Upstate
Biotechnology Inc.). Washes of the protein A Sepharose
complexes were
performed in the same buffer. Supernatant from
the immunoprecipitation
corresponded to the receptor-depleted
membrane fraction whereas the
bead pellet, further washed in the
same buffer, provided the sample of
immunoprecipitated
receptor.
Cloning of histidine-tagged STATs for expression in E. coli: purification of the recombinant STATs.
Stat1 and Stat3
cDNAs were cloned downstream of a decahistidine tag in a pET-19b vector
(Novagen). For Stat1, an additional hemagglutinin (Lerner) tag was
inserted between the polyhistidine tag and the STAT ATG. Proteins were
produced in E. coli and purified under native conditions on
a nickel resin. For that purpose, BL21 strains expressing the
recombinant STATs were grown in Luria-Bertani medium to an optical
density at 600 nm of 0.8 and induced with 1 mM
isopropyl--D-thiogalactopyranoside (IPTG) for 3 h.
Cells were lysed by sonication in a buffer containing 50 mM
Na-phosphate (pH 8.0); 300 mM NaCl; 0.25% Triton; 1-µg/ml
concentrations of leupeptin, aprotinin, and pepstatin; 1 mM PMSF; and 3 mM -mercaptoethanol. The sonication supernatant was adjusted to a
final concentration of 40 mM imidazole and mixed with 200 µl of
nickel packed beads for 30 min at 4°C. The beads were loaded onto a
column and washed with 25 bed volumes of buffer containing 50 mM
Na-phosphate (pH 8.0); 300 mM NaCl; 1-µg/ml concentrations of
leupeptin, aprotinin, and pepstatin; 1 mM PMSF; 3 mM
-mercaptoethanol; 40 mM imidazole; and 10% glycerol. Elution of
recombinant Stat1 and Stat3 was achieved with 120 mM and 200 mM
imidazole, respectively. Stat3 fractions were immediately diluted
twofold in washing buffer devoid of NaCl and imidazole. Protein
fractions were kept at 
70°C.
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RESULTS |
PDGF-dependent activation of Stat1 and Stat3 in vitro.
Stat1
and Stat3 are activated by the PDGF receptor in vivo (22,
23). When assayed by mobility shift assay on a DNA probe derived from the c-fos SIE (23), activated STATs form
three specific complexes, termed SIF-A, SIF-B, and SIF-C,
which correspond respectively to the binding of
Stat3 homodimers, Stat1/Stat3 heterodimers, and Stat1
homodimers. We showed previously that in 2fTGH.PS1 cells (human
fibrosarcoma cells engineered to express the PDGF- receptor), binding of PDGF-BB to its receptor leads to the activation of Stat1 and Stat3 and the formation of all three SIF complexes
(22).
To examine the biochemical mechanism of STAT activation by the PDGF
receptor in 2fTGH.PS1 cells, we extended a previous study
(
16) to develop a cell-free system composed of
membrane and
cytosol fractions derived from these cells.
Addition of PDGF,
magnesium, and ATP to reactions composed
of 2fTGH.PS1 membranes
and cytosol followed by incubation at
30°C for 30 min led to activation
of STAT proteins and the appearance
of SIF complexes on a mobility
shift gel (Fig.
1A, lanes 1 to 8). Activation required
both membrane
and cytosol fractions and was dependent upon the addition
of both
ATP and PDGF (Fig.
1A and data not shown). These reaction
conditions
favored the activation of Stat1 over Stat3, as SIF-C, a
Stat1
homodimer, was the primary product (lanes 6 and 8). This
observation
contrasts with the pattern observed following PDGF
stimulation
of the same cells in vivo, which leads primarily to
activation
of Stat3 and formation of the SIF-A complex (
22).
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FIG. 1.
(A) PDGF-dependent activation of Stat1 and Stat3 in
vitro. Detergent-solubilized membrane proteins from 2fTGH.PS1 cells,
which overexpress the PDGF- receptor, were mixed with cytosolic
extracts from 2fTGH.PS1 or HeLa cells. The proteins were subjected to
an in vitro kinase reaction in the presence of ATP and magnesium, and
the activated STATs were analyzed by mobility shift assay using the
high-affinity SIEm67 32P-labeled probe. Lanes: 1 and 2, no
cytosolic extract; 3 to 8, 2fTGH.PS1 cytosol; 9 to 14, HeLa cytosol.
The kinase reactions were done with (+) or without () membranes (mb)
as indicated and in the presence (+) or absence () of PDGF as
indicated. The three specific complexes SIF-A, SIF-B, and SIF-C are
indicated at right. (B) 2fTGH.PS1 and HeLa cells express similar
concentrations of Stat1 as well as of Stat3 proteins. Equal amounts (10 and 30 µg, respectively) of cytosolic extracts from 2fTGH.PS1 or HeLa
cells were analyzed by Western blotting using antibodies directed
against either Stat1 or Stat3. The STAT protein recognized by the
antibodies is indicated.
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When cytosol from HeLa cells was substituted for 2fTGH.PS1 cytosol, the
pattern of STAT activation changed. With HeLa cytosol,
significant
amounts of the SIF-A and -B complexes were formed,
indicating efficient
activation of Stat3 as well as Stat1 (Fig.
1A, lane 14). The ability of
HeLa cytosol to support the activation
of Stat3 to a significantly
greater extent than 2fTGH.PS1 cytosol
could be due to a correspondingly
higher concentration of Stat3
protein in the HeLa cytosol fraction. To
test this idea, we compared
the concentrations of Stat1 and Stat3 in
2fTGH.PS1 and HeLa cytosol
by Western blotting (Fig.
1B). No
significant differences were
observed for either protein, suggesting
that another difference
in the constitution of the HeLa cytosol
fraction accounts for
its ability to support Stat3 activation in
vitro.
Thus, this cell-free system is able to support the activation of both
Stat1 and Stat3 by PDGF. Stat1 was activated with similar
efficiency by
using cytosolic fractions from either 2fTGH.PS1
or HeLa cells. In
contrast, only HeLa cell extracts supported
the activation of Stat3.
These observations suggest that the biochemical
mechanisms of
activation of Stat1 and Stat3 may
differ.
In vitro activation of a purified recombinant Stat1 protein.
To begin to dissect the mechanisms of activation of Stat1 and Stat3 in
vitro, we produced recombinant Stat1 and Stat3 in E. coli. Both proteins carried N-terminal histidine tags, allowing them to be purified on nickel resin, leading to the protein
preparations shown in Fig. 2. The
recombinant Stat1 protein also carried an epitope tag immediately
C-terminal to the histidine tag. A Stat3 protein containing the epitope
tag could not be produced.
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FIG. 2.
Purification of Stat1 and Stat3. Histidine-tagged Stat1
and Stat3 proteins were expressed in E. coli and
purified on nickel. The purified proteins were analyzed by
polyacrylamide gel electrophoresis and revealed by Coomassie staining.
Stat3 harbors the histidine tag at its N terminus (HSTAT3) while Stat1
contains an additional hemagglutinin tag between the polyhistidine
sequence and the N terminus of the protein (HLSTAT1). The molecular
weight (MW) markers are indicated (in thousands) on the left of the
gel.
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To determine whether purified recombinant Stat1 (HLSTAT1)
could substitute for the cytosolic fraction in our
PDGF-dependent
system, we added HLSTAT1 to a membrane
fraction from 2fTGH.PS1
cells. The addition of PDGF to reactions
composed of the membrane
fraction and purified HLSTAT1 led to the
appearance of a SIF-C
complex (Fig.
3A,
lane 5). This complex was not observed when
HLSTAT1 was omitted
from the reaction (lane 2). Furthermore, the
addition of a monoclonal
antibody that recognizes an N-terminal
epitope tag incorporated into
HLSTAT1 shifted the mobility of
the induced SIF-C complex (lane 6).
Thus, this complex contains
the recombinant Stat1 protein, and these
data suggest that the
2fTGH.PS1 membrane fraction is sufficient to
support the activation
of Stat1 in vitro.
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FIG. 3.
(A) In vitro activation of the purified Stat protein.
The purified Stat1 protein was mixed with 2fTGH.PS1 membranes (mb),
activated in vitro, and tested by EMSA on an SIE probe (lanes 4 to 6).
Lanes 1 to 3, mock reactions with membranes only. The kinase reactions
were done in the presence (+) or absence () of PDGF. The addition of
the 12CA5 monoclonal antibody during the gel shift reaction is
indicated by an asterisk. The SIF-C complex corresponding to the
binding of Stat1 to the SIE site is indicated at the right. (B) The
PDGF-dependent activation of Stat1 is inhibited by cytosolic proteins.
The purified HLSTAT1 protein was mixed with increasing
concentrations of U3A cytosolic extracts and subjected to the in vitro
kinase reaction using 2fTGH.PS1 membrane fractions as the source of
PDGF- receptor. As a control, U3A cytosolic proteins (25 µg) were
tested in the kinase reaction in the absence of recombinant Stat1
protein (lanes 1 and 2). The kinase reactions were done in the absence
() or presence (+) of PDGF. The SIF-C complex is indicated.
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To determine whether additional activities in the cytosol might also be
required for Stat1 activation by PDGF, we added cytosolic
extracts from
U3A cells, a derivative of 2fTGH that lacks endogenous
Stat1
(
13). This fraction failed to stimulate PDGF induction
of
HLSTAT1 DNA-binding activity (Fig.
3B) or phosphorylation of
HLSTAT1 on tyrosine-701 (Fig.
4). Rather, it exhibited
dose-dependent
inhibition of HLSTAT1 activation. Taken together,
these observations
suggest that membrane-associated proteins from
2fTGH.PS1 cells
are sufficient for activation of Stat1 by PDGFR in
vitro and that
no additional activities from the cytosol are required.
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FIG. 4.
Stat1 tyrosine phosphorylation and DNA binding activity
are both inhibited by cytosolic proteins. The purified HLSTAT1
protein was subjected to the in vitro kinase reaction using 2fTGH.PS1
membranes (40 µg), in the absence () or presence (+) of U3A
cytosolic extract (100 µg). Following the kinase reaction, half of
the reaction mixture was analyzed by Western blotting using Stat1
phosphotyrosine (Y701) antibodies (A), the second half of the reaction
mixture was analyzed by EMSA on an SIE probe (B). The phosphorylated
Stat1 protein is indicated. Reprobing of the blot with an
antihemagglutinin antibody (12CA5) shows equal Stat1 concentrations in
the various lanes.
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To determine whether membrane proteins other than PDGFR
itself are required for activation of HLSTAT1 by PDGF, we
purified
PDGFR from detergent-solubilized 2fTGH.PS1 membrane fractions
by specific immunoprecipitation. Incubation of HLSTAT1 with
immunopurified
PDGFR led to ATP-dependent and partially
ligand-dependent Stat1
activation (Fig.
5, lanes 4 to 6). The background
activation of
Stat1 in the absence of PDGF (lane 5) could be due to
cross-linking
of the PDGFR by antibody. In contrast, incubation of
HLSTAT1 with
immune complexes obtained with normal rabbit serum did
not lead
to Stat1 activation (lanes 13 to 15). HLSTAT1 was not
activated
by a membrane fraction that had been depleted of PDGFR
with the
receptor antibody (lanes 7 to 9), indicating that PDGFR
was required
for activation of Stat1 by PDGF, whereas activation
by the membrane
fraction was not affected by depletion with control
serum (lanes
16 to 18). Furthermore, activation of HLSTAT1 by
immunopurified
PDGFR was not further augmented by the addition of the
receptor-depleted
membrane fraction (lanes 10 to 12). These
observations suggest
that purified PDGFR is sufficient for the
activation of HLSTAT1
in vitro. We cannot formally rule out the
possibility that another
factor tightly associated with the receptor
might act as an intermediate
in Stat1 activation by PDGFR. However, any
such protein would
have to be present in excess in the receptor
complex, since supplementation
with the receptor-depleted membrane
fraction did not further increase
HLSTAT1 activation. We
provisionally conclude that PDGFR is both
necessary and sufficient for
Stat1 activation in vitro.
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FIG. 5.
Activation of Stat1 protein by the immunoprecipitated
PDGF- receptor. The PDGF- receptor was immunoprecipitated from
detergent-solubilized membranes of 2fTGH.PS1 cells. The purified Stat1
was incubated in the in vitro kinase reaction with the
immunoprecipitated PDGFR [IP (R); lanes 4 to 6], the membrane
fraction depleted of PDGFR [Sup (R); lanes 7 to 9] or a mixture of
the two [(IP + Sup) (R); lanes 10 to 12] and tested by EMSA on
an SIE probe. In the mock immunoprecipitations [IP (C); lanes 13 to
15] or depletions [Sup (C); lanes 16 to 18], normal rabbit serum was
used instead of specific anti-PDGFR antibodies. Lanes 1 to 3, control
reactions where Stat1 was incubated without membrane proteins. The
kinase reactions were done in the presence (+) or absence () of PDGF
as well as of ATP/magnesium. The SIF-C complex corresponding to Stat1
binding to the SIE site is indicated.
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In vitro activation of Stat3 requires additional cytoplasmic
factors.
We performed a similar analysis using purified
recombinant Stat3 (HSTAT3). In contrast to our observations with
Stat1, the 2fTGH.PS1 membrane fraction alone was insufficient to
support PDGF-dependent activation of Stat3 (Fig.
6A, lane 4). However, the addition of U3A
cytosol, which was unable on its own to support SIF-A activation (lane
2) and inhibited activation of Stat1, led to a dose-dependent induction
of the SIF-A complex, indicating activation of HSTAT3 (lanes 6 and 8).
Thus, the combination of 2fTGH.PS1 membranes and U3A cytosol was
required to reconstitute PDGF-dependent activation of purified
recombinant Stat3 in vitro.
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FIG. 6.
(A) In vitro activation of Stat3 by the PDGF receptor
requires cytosolic proteins. The purified Stat3 protein (HSTAT3; lanes
3 to 8) or a mixture of Stat1 and Stat3 (HLSTAT1 + HSTAT3;
lanes 9 to 14) were incubated with increasing concentrations of U3A
cytosol, activated in vitro, and tested by EMSA on an SIE probe. As a
control, the kinase reaction was done with U3A cytosol (25 µg), in
the absence of recombinant STATs (lanes 1 and 2). Kinase reactions were
done in the presence (+) or absence () of PDGF. The three specific
complexes SIF-A, SIF-B, and SIF-C are indicated. (B) The SIF-A complex
generated in vitro corresponds to the activation of the recombinant
Stat3 protein. HSTAT3 was incubated with U3A cytosol for the kinase
assay and tested by EMSA (lanes 4 to 6). Control reactions were
performed in the absence of recombinant Stat3 (lanes 1 to 3). In lanes
3 and 6, protein extracts obtained by the kinase reactions were
depleted on nickel resin of histidine-containing proteins prior to the
EMSA. Depletion on nickel resin is indicated by an asterisk. The SIF-A
complex obtained in the presence (+) of PDGF is indicated. The left
panel shows a longer exposure of the gel (lanes 1L to
3L).
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Because the addition of U3A cytosol appeared to have opposite effects
on the activation of recombinant Stat1 and Stat3, respectively,
we
added U3A cytosol to a reaction containing a mixture of HLSTAT1
and
HSTAT3. In the absence of cytosol, we mainly observed the
induction of
SIF-C (lane 10), whereas the addition of U3A cytosol
led to the
disappearance of SIF-C with the concomitant appearance
of SIF-A (lanes
12 and 14). This observation reinforces the conclusion
that activation
of Stat3 but not Stat1 requires activities present
in U3A cytosol and,
therefore, that the mechanisms of activation
of Stat1 and Stat3 must
differ.
Although U3A cytosol without supplementation with recombinant Stat3
protein was unable to support SIF-A activation (Fig.
6A,
lane 2;
see also Fig.
6B, lane 2), we performed an additional
experiment
to verify that the SIF-A activity observed in the presence
of U3A
cytosol and HSTAT3 was indeed derived from the recombinant
protein. An
in vitro reaction containing both cytosol and HSTAT3
led to the
appearance of the expected SIF-A complex (Fig.
6B,
lane 5). If this
activity contains the histidine-tagged recombinant
protein, we expected
to be able to deplete this activity with
nickel resin. Therefore,
following completion of the activation
reaction, but prior to mobility
shift analysis, the reaction was
incubated with nickel resin, and the
unbound material was analyzed
by mobility shift assay. Incubation with
nickel resin led to complete
disappearance of SIF-A (lane 6),
consistent with the idea that
the SIF-A activity generated in this
reaction was composed of
recombinant
protein.
To verify that the effect of the nickel resin was specific, we
performed a parallel depletion on U3A cytosol alone. Although
SIF-A activation by cytosol alone was not detectable at the same
exposure level used to visualize activation of the recombinant
protein, exposure of the same gel for several days allowed
visualization
of a weak SIF-A band derived from endogenous U3A Stat3
protein
(lane 2
L). This band was not affected by incubation
with nickel
resin (lane 3
L). Thus, we conclude that
membranes and cytosol
are necessary and sufficient to activate purified
recombinant
Stat3.
JAK proteins are required for the activation of Stat3 by the PDGF
receptor.
Our results suggest that activation of Stat3 by PDGFR in
vitro requires proteins in the cytosolic fraction. One obvious
candidate is the JAK family. To determine whether JAK proteins were
required for the activity present in U3A cytosol, we treated the
cytosol with a cocktail of antibodies specific for JAK1, JAK2, and
Tyk2, respectively, with the goal of depleting the cytosol of all JAK activity. We conducted in vitro activation assays composed of 2fTGH.PS1
membranes, a mixture of recombinant Stat1 and Stat3, and U3A cytosol
depleted with various antibodies. Treatment of U3A cytosol with normal
rabbit serum led to a pattern of SIF activation similar to that seen
with untreated cytosol (compare Fig. 7A, lane 2, to Fig. 6A, lane 14)
the predominant activities observed were
SIF-A and -B. Treatment of the cytosol with antibody to Erk2 also did
not affect induction of SIF-A and -B (Fig. 7A, lane 6). In contrast,
when U3A cytosol was depleted with the cocktail of anti-JAK antibodies,
activation of SIF-A and -B was significantly reduced (lane 4). This
observation suggests that JAK proteins are required for U3A cytosol to
support Stat3 activation.
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FIG. 7.
(A) JAKs are required for Stat3 activation. A mixture of
purified Stat1 and Stat3 recombinant proteins was incubated and
activated in vitro with U3A cytosolic extracts that had been previously
incubated with a cocktail of antibodies (Ab) to JAKs (lanes 3 and 4),
with antibodies to ERK2 (lanes 5 and 6), or, as a control, with normal
rabbit serum (NRS) (lanes 1 and 2). The activated STATs were then
tested by EMSA on an SIE probe. The kinase reactions were done in the
presence (+) or absence () of PDGF. The three specific complexes
SIF-A, SIF-B, and SIF-C are indicated. (B) Individual JAKs contribute
to Stat3 activation by the PDGF- receptor. The purified Stat3
protein was incubated with U3A cytosol that had been previously
depleted using antibodies to JAK1 (lanes 3 and 4), Tyk2 (lanes 5 and
6), a mixture of these two antibodies (lanes 7 and 8), or, as a
control, with normal rabbit serum (lanes 1 and 2). The kinase reactions
were done in the presence (+) or absence () of PDGF. The SIF-A
complex obtained by EMSA on an SIE probe is indicated.
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To confirm that JAKs were required for Stat3 activation in vitro, we
repeated the depletion assay with antibodies against
individual JAKs.
Specific depletion of the U3A cytosol with antibodies
to JAK1 (Fig.
7B,
lane 4), Tyk2 (lane 6), and JAK2 (data not shown)
each led to a partial
inhibition of SIF-A induction. The effect
of each antibody appeared to
be additive, as seen by the results
shown in lane 8. Thus, we believe
that the observed requirement
for JAKs can be satisfied by any of the
JAK
proteins.
In summary, we find that the biochemical mechanisms of activation of
Stat1 and Stat3 by PDGFR are distinct. Stat1 activation
does not
require any activities present in the cytosol. Indeed,
the purified
PDGFR appears to be sufficient for Stat1 activation.
In contrast, Stat3
activation requires activities present in the
cytosol. One of those
required activities is supplied redundantly
by the JAK
proteins.
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DISCUSSION |
We have developed a cell-free system that reproduces
PDGF-dependent activation of Stat1 and Stat3. This system is composed of a membrane fraction derived from a cell line that overexpresses PDGFR (22) and was supplemented with either recombinant
purified STAT proteins or with complex cytosolic fractions from tissue culture cells. Analysis of this system revealed unexpected differences in the mechanisms of activation of the two STAT proteins. Activation of
Stat1 could be reconstituted with immunopurified PDGFR and purified recombinant Stat1. The addition of neither membrane nor cytosol fractions augmented Stat1 activation, indicating that PDGFR alone is probably sufficient for Stat1 activation in vitro. Although membrane fractions were prepared from extensively starved 2fTGH.PS1 cells to minimize any preexisting association of PDGFR with
signaling intermediates, we cannot unambiguously rule out a requirement
for an additional receptor-associated factor. Nevertheless, we favor
the hypothesis that Stat1 is a direct substrate for PDGFR kinase.
We found that cytosolic fractions inhibited the activation of Stat1 by
purified PDGFR. Several activities in the cytosol could be responsible
for the observed inhibition of Stat1 tyrosine-701 phosphorylation. The
cytosol could contain high levels of a phosphatase that reverses Stat1
phosphorylation. A second possibility is that the cytosol provides
SH2-containing proteins that compete with Stat1 for its putative
binding sites on the PDGFR, as has been shown for other STATs (1,
6, 14, 20).
In contrast to the apparently direct interaction between PDGFR and
Stat1, PDGF-dependent activation of Stat3 displays a strong requirement
for the cytosolic fraction. Depletion of JAK proteins from the cytosol
abolishes this activity, suggesting that JAKs are required for
activation of Stat3 by PDGFR. We have previously shown that all three
ubiquitously expressed JAKs are associated with the activated PDGFR
(22), although their role in PDGF signaling was not
established. JAKs could be performing several functions in linking
PDGFR to Stat3 activation in our cell-free system. First,
receptor-associated JAKs could phosphorylate the receptor to generate
Stat3-specific recruitment sites that are not phosphorylated by the
receptor itself. Second, the JAKs could directly phosphorylate Stat3 itself, an activity presumably not enabled in PDGFR. Third, JAKs could provide Stat3 recruitment sites in the receptor complex, as
proposed for Jak2 in response to growth hormone receptor activation (19).
Although PDGF treatment of intact 2fTGH.PS1 cells leads to activation
of both Stat1 and Stat3 in roughly equal measure, a cell-free system
composed of membrane and cytosol fractions from the same cells
activates Stat1 almost exclusively. What accounts for the selective
loss of Stat3 activation in the cell-free system? One simple
explanation is that Stat1 activation is presumably a two-component
reaction, comprising just PDGFR and Stat1, whereas Stat3
activation requires at least a third component present in the cytosol.
Thus, Stat3 activation, which requires the association of at least
three factors, should be much more sensitive to changes in the
concentration of the reactants. Since the cell-free reaction is
probably substantially more dilute with respect to the reactants than
intact cells, Stat3 activation would be preferentially lost in vitro.
That Stat3 activation is not substantially lost when HeLa cytosol is
used in place of 2fTGH.PS1 cytosol suggests that one component of the
Stat3 activation reaction, presumably the JAKs, is present at either a
higher concentration or higher specific activity in the HeLa fraction.
Finally, we showed previously that PDGF induced the phosphorylation of
all three JAKs in 2fTGH.PS1 cells but that no single JAK was uniquely
required for STAT activation by PDGF (22). We reached this
conclusion based on the use of cell lines that lacked individual JAKs.
Because we had no cell lines lacking all of the JAKs, we could not rule
out the possibility that the three JAKs present in these cells were
functionally redundant for STAT activation but that at least one was
required. The biochemical experiments we describe here allowed us to
address this possibility, since we could deplete the JAKs both
individually and in combination. Indeed, we found that depletion of all
three JAK proteins from the cytosolic fraction substantially eliminated
Stat3 activation in vitro, whereas depletion of individual JAKs
yielded intermediate effects. Thus, we believe that each JAK is
independently capable of mediating Stat3 activation by PDGFR.
| |
ACKNOWLEDGMENTS |
We thank Theresa Nahreini for help with protein expression and
Mandy Cunningham for manuscript preparation.
This work was supported by Public Health Service grant CA45642 from the
National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Present address: 550 Chestnut
St., Waban, MA 02468. Phone: (617) 244-3248. Fax: (617) 244-6249. E-mail: gilman{at}akamail.com.
Present address: IGM-CNRS UMR 5535, 34293 Montpellier Cedex 5, France.
| |
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Molecular and Cellular Biology, May 1999, p. 3727-3735, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
