From the Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland
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ABSTRACT |
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Epidermal growth factor (EGF) binding to its
receptor, ErbB1, triggers various signal transduction pathways, one of
which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB
receptors have not been explored. In this report we show that ErbB2,
ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF.
However, neu differentiation factor-induced heterodimers of ErbB2 and
ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were
constitutively complexed with ErbB1 and rapidly phosphorylated on
tyrosine in response to EGF. Neither mutation of the conserved tyrosine
residue (Tyr694) nor inactivation of the Stat5a SH2
domain disrupted this association. However, an intact SH2 domain was
necessary for EGF-induced Stat5a phosphorylation. In contrast to
prolactin, which induced only Tyr694 phosphorylation of
Stat5a, EGF promoted phosphorylation on Tyr694 and
additional tyrosine residue(s). Janus kinases (Jaks) were also
constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that
EGF- and neu differentiation factor-induced Stat activation are
dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was
rapidly recruited to Stat/ErbB receptor complexes. Pharmacological Src
kinase inhibitors and a dominant negative c-Src ablated both Stat and
Jak tyrosine phosphorylation. However, dominant negative Jaks did not
affect EGF-induced Stat phosphorylation. Taken together, the
experiments establish two independent roles for Src kinases: (i) key
molecules in ErbB receptor-mediated Stat signaling and (ii) potential
upstream regulators of Jak kinases.
The ErbB family of receptor tyrosine kinases, consisting of the
epidermal growth factor
(EGF)1 receptor ErbB1,
ErbB2/Neu, ErbB3, and ErbB4, binds a large group of ligands, the
EGF-related peptides (1, 2). Upon ligand-induced dimerization, the
receptors autophosphorylate on specific tyrosine residues in their
cytoplasmic tails. These residues provide docking sites for
phosphotyrosine binding, cytoplasmic signaling molecules that activate
numerous intracellular signaling pathways. In some cases, for example
the mitogen-activated protein (MAP) kinase pathway, the important
intermediates in the signaling cascade have been well described (3).
Receptor tyrosine kinase-mediated activation of signal transducer and
activators of transcription (Stat) proteins, however, is less well understood.
Stats were initially identified as a family of transcription factors
activated by engagement of interferon (IFN) and cytokine receptors,
which lack intrinsic kinase activity and depend upon the
receptor-associated Janus kinases (Jaks) to transduce signals. Cytokine
receptor oligomerization brings the Jaks into juxtaposition, leading to
their cross-phosphorylation and activation. Jaks in turn phosphorylate
receptors on tyrosine residues, thereby providing docking sites for
downstream signaling proteins. The Stats that are recruited to the
Jak-receptor complex via their Src homology 2 (SH2) domains are
phosphorylated on a conserved tyrosine residue in the C terminus. This
phosphorylation is sufficient for Stat dimerization, nuclear
translocation, and binding to specific DNA sequences, affecting the
expression of multiple target genes (reviewed in Refs. 4 and 5).
Ligand-activated platelet-derived growth factor and EGF receptors
stimulate Stat phosphorylation and DNA binding activity (reviewed in
Ref. 6). The intrinsic kinase activity of these receptors, but not that
of Jaks, appears to be required for Stat activation (7, 8). Immune
complex-purified ErbB1 was shown to phosphorylate Stat1 on the
conserved tyrosine residue in the C terminus (9), and the
baculovirus-expressed kinase domain of ErbB1 phosphorylated Stat3
in vitro (10). Whether or not additional proteins are
involved in receptor tyrosine kinase-mediated Stat activation in
vivo has not been resolved. In addition, the role that other ErbB
family members play in Stat activation has not been analyzed.
ErbB2, which has no direct ligand but heterodimerizes with all other
family members upon ligand binding (11, 12), as well as ErbB3 and
ErbB4, which both bind neu differentiation factor (NDF), were tested
for their ability to activate Stats. We used NIH3T3 sublines expressing
pairs of ErbB receptors (13) to examine dimerization-dependent differences in Stat activation.
ErbB2, which is known to potentiate EGF- and NDF-induced MAP and S6
kinase activation (14), did not affect EGF-induced Stat activation, suggesting that ErbB1 expression is sufficient for this activity. Interestingly, NDF treatment of cells coexpressing ErbB2 and ErbB4, but
not ErbB2 and ErbB3, led to Stat activation. This is a novel signaling
property specific for the heterodimer because homodimers of either
receptor were inefficient at activating Stat5.
In addition to receptor tyrosine kinases and Jaks, v-Src and v-Abl
kinases have been implicated in Stat signaling (15-17). Transformation
of cells by these oncoproteins induces constitutive activation of Stat
transcription factors. Because the c-Src tyrosine kinase lies
downstream of ErbB receptors (18-22), we examined the role of Src
kinases in ErbB receptor-mediated Stat activation. Using
pharmacological Src kinase inhibitors and dominant negative (DN) c-Src
and Jak expression vectors, we provide evidence that Src but not Jak
kinase activity is required for ErbB receptor-mediated Stat activation.
Thus, Stat activation by ErbB receptors proceeds via a mechanism
distinct from the classical Jak-dependent pathway described
for IFN and cytokine receptor signaling.
Antibodies, Growth Factors, and Inhibitors--
The antibodies
used were: ErbB1-specific monoclonal antibodies (mAbs) EGFR1 and 528 and rabbit polyclonal antibody 1005 (Santa Cruz Biotechnology),
ErbB2-specific polyclonal rabbit antiserum 21N (23), Stat1-specific
antibody E23 and Stat3-specific antibody H190 (Santa Cruz
Biotechnology), Stat5a and Stat5b polyclonal anti-peptide antisera
(24), Jak2-specific polyclonal antibody C20 (Santa Cruz Biotechnology),
Jak1- and Jak2-specific rabbit antisera (UBI), Src-specific mAb 327 (provided by K. Ballmer-Hofer (Paul Scherrer Institute, Villigen,
Switzerland) and purchased from Calbiochem as Ab-1),
phosphotyrosine-specific mAb (25) and hemagglutinin (HA)
epitope-specific mAb (26). Growth factors and hormones used were:
recombinant human EGF (Sigma); recombinant human heparin-binding EGF
and betacellulin (R&D Systems), recombinant human NDF (initially
provided by Amgen and then purchased from Neomarkers), dexamethasone
(Sigma), insulin (Sigma), and ovine prolactin (luteotropic hormone)
(Sigma). The Src kinase inhibitors used were: PP1 (Calbiochem) (27) and
CGP77675 (J. Green, Novartis) (28).
Cell Lines and Cell Culture--
NIH3T3 derivative cell lines
ectopically expressing human ErbB receptors (13) and the vulval
carcinoma cell line A431 were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum. NE1/2/3 and NE1/2/4
cells were generated by stable introduction of pBabe-based retroviruses
(29) encoding human ErbB2 into NE1/3 and NE1/4 cells, respectively.
Prior to growth factor stimulation, cells were starved for 18 h in
serum-free medium (Dulbecco's modified Eagle's medium containing 1 mg/ml fetuin (Sigma) and 10 µg/ml transferrin (Sigma)). HC11 mammary epithelial cells were grown to confluency and maintained in RPMI 1640 supplemented with 10% fetal calf serum, 10 ng/ml EGF, and 5 µg/ml
insulin. The cells were starved for 12 h in serum-free medium
prior to induction for 30 min with the lactogenic hormones (10 Hypertonic Lysis of Cells, Nuclear Extracts, and Electrophoretic
Mobility Shift Assay (EMSA)--
Cells were scraped into cold
hypertonic buffer (400 mM KCl, 10 mM
NaH2PO4, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium fluoride, 50 µM
Immunoprecipitations and Western Blotting--
Whole cell
extracts (WCE) were obtained by solubilizing cells in Nonidet P-40
extraction buffer (50 mM Tris, pH 7.5, 5 mM EGTA, 120 mM NaCl, 1% Nonidet P-40, 2 mM
sodium orthovanadate, 50 mM sodium fluoride, 20 mM Src Kinase Assays--
Cells were lysed in Nonidet P-40
extraction buffer as described under "Immunoprecipitations and
Western Blotting." After preclearing of lysates with protein
G-Sepharose (Sigma), c-Src was immunoprecipitated. Immunoprecipitates
were washed twice with extraction buffer and twice with kinase buffer
(50 mM Tris, pH 7.5, 5 mM MgCl2).
The immune complexes were incubated with 5 µg of acid-denatured
enolase (Sigma) for 10 min at 30 °C in 30 µl of kinase buffer
containing 5 µM ATP and 5 µCi
[ Plasmids and Transient Transfections--
An HA-tagged wild type
(WT) Stat5a cDNA was cloned into the mammalian expression vector
pcDNA3.1. Mutations (Y694F and R618L) were introduced by
site-directed mutagenesis. The expression plasmid encoding WT c-Src was
provided by G. Superti-Furga (EMBL, Heidelberg); the plasmid encoding
DN c-Src was provided by S. Courtneidge (Sugen, Inc.). DN Src has a
K298M substitution in the ATP binding site and a Y530F substitution in
the regulatory tyrosine, thereby rendering the protein in an open
conformation that enables binding of substrates (32). DN Jak1 and Jak2
expression vectors were provided by O. Silvennoinen (University of
Tampere, Finland). The expression vector encoding the prolactin
receptor was provided by B. Groner (Georg Speyer Haus, Frankfurt).
Plasmids were introduced into cells using Fugene transfection reagent
(Roche Molecular Biochemicals) according to the manufacturer's
protocol. Eight hours posttransfection, cells were starved for 16 h in serum-free medium prior to stimulation with growth factors.
ErbB Receptor-mediated Activation of the Stat Signaling
Pathway--
It has previously been shown that signaling through ErbB1
leads to phosphorylation and activation of Stat1, Stat3, and Stat5 (33-36). In this study we investigated whether Stat proteins are downstream targets of other ErbB receptors. To examine the contribution of ErbB2, ErbB3, and ErbB4 to EGF-induced Stat activation and the
ability of the ErbB3/ErbB4 ligand, NDF, to activate these proteins, we
used NIH3T3 sublines expressing defined combinations of ErbB receptors
as a model cellular system (13). NIH3T3 cells lack ErbB1, ErbB3, and
ErbB4 and have very low levels of endogenous ErbB2. Ectopic expression
of human ErbB1 and ErbB2 in NIH3T3 cells gave rise to the NE1 and NE2
clones, respectively. NE1-derived cell lines coexpressing ErbB2, ErbB3,
or ErbB4 were designated NE1/2, NE1/3, and NE1/4, respectively.
Likewise, NE2-derived cell lines coexpressing ErbB3 or ErbB4 were
designated NE2/3 and NE2/4, respectively (13). In NE1 cells, EGF at 1 nM was efficient at activating the Stat5 isoforms a and b
as measured by their tyrosine phosphorylation (not shown). In addition
to homodimers of ErbB1, EGF can induce heterodimers of ErbB1 with
ErbB2, ErbB3, or ErbB4 (11). However, these heterodimers appeared not
to contribute to Stat5b activation because phosphorylation of Stat5b in
the NE1 derivatives was unaffected by coexpression of ErbB2, ErbB3, or
ErbB4 (Fig. 1A). Because ErbB2
enhances EGF-mediated activation of ErbB3 and ErbB4 (11) we introduced
ErbB2 into NE1/3 and NE1/4 cells, thereby generating the NE1/2/3 and
NE1/2/4 cell lines, respectively. Expression of ErbB2, however, failed
to potentiate EGF-induced activation of Stat5b (Fig. 1A).
These results suggest that ErbB1 expression is sufficient for maximal
EGF-induced Stat5 phosphorylation.
NDF, which binds and activates ErbB2/ErbB3 heterodimers in NE2/3 cells,
did not promote Stat5b phosphorylation (Fig. 1B). In NE2/4
cells, however, in which NDF activates ErbB4 homodimers and ErbB2/4
heterodimers, a strong increase of Stat5b phosphotyrosine was observed
(Fig. 1B). In NDF-treated NE1/4 cells, in which mainly ErbB4
homodimers are activated (13), there was basically no increase in
Stat5b phosphorylation (Fig. 1B). The mAb FRP5, which induces homodimers of ErbB2 and activates MAP and S6 kinases (37), also
did not promote phosphorylation of Stat5b (not shown). Taken together,
the results suggest that coexpression of ErbB2 and ErbB4 is required
for Stat5b activation in response to NDF. The very weak Stat5b
phosphorylation seen in NE1/4 cells is most likely because of
heterodimers of ErbB4 with the low levels of endogenous ErbB2.
The ErbB1-overexpressing A431 carcinoma cell line also expresses ErbB2
and ErbB3, but lacks ErbB4. Treatment of these cells with the ErbB1
agonists EGF, heparin-binding EGF, and betacellulin led to Stat5b
phosphorylation (Fig. 1C). NDF treatment, however, failed to
induce Stat5b tyrosine phosphorylation, which is in accordance with the
results obtained with the NIH derivatives.
EGF-induced Activation of Stat1, Stat3, and Stat5 and Their
Constitutive Association with ErbB1--
A431 cells express Stat1,
Stat3, and Stat5b and low levels of Stat5a. In these cells, EGF
treatment leads to activation of Stat1 and Stat3 (33, 36) and of both
Stat5 proteins (Fig. 2). We analyzed the
kinetics of EGF-induced Stat activation in these cells by treating them
with growth factor for various times ranging from 5 min to 2 h.
EGF promoted a rapid increase in the tyrosine phosphorylation of all
Stat proteins (Fig. 2). Activation of
Stat3 was the most transient (Fig. 2B), and Stat5b
phosphorylation was the strongest and most sustained (Fig.
2D).
In cytokine and IFN signaling, Stat transcription factors are recruited
to ligand-activated receptors. We, therefore, asked whether there was a
physical interaction of Stat proteins with ErbB1. Probing of ErbB1
immunoprecipitates from A431 cells with Stat-specific antibodies
demonstrated that there was stable EGF-independent association of Stat1
(mainly the p91 isoform) and Stat3 with the receptor (Fig.
3, A and B).
Detection of endogenous Stat5 in immunoprecipitates of ErbB1 proved to
be difficult. However, following transient expression of HA-tagged
Stat5a in A431 cells, ErbB1 was readily detected in HA-Stat5a
immunoprecipitates (Fig. 3C). Reprobing of the membrane
using HA-specific mAb showed that equal amounts of HA-Stat5a were
expressed and immunoprecipitated (Fig. 3C). We confirmed by
probing with Stat5a-specific serum that the top band comigrates with
Stat5a (not shown); the lower band is nonspecific. In summary, all Stat
proteins were found complexed with ErbB1 even in the absence of growth
factor. Although ErbB1 from nonstimulated A431 cells contains high
basal phosphotyrosine (not shown), suggesting some degree of
activation, the phosphorylation of associated Stats was strictly
ligand-dependent (Fig. 2).
EGF Promotes Phosphorylation of Stat5a on Additional
Tyrosine(s)--
To test which regions of Stat proteins are essential
for their interaction with and activation by ErbB receptors, we
constructed HA-tagged mutants of Stat5a with (i) a Y694F substitution
in the C-terminal consensus tyrosine residue (Tyr mutant) and (ii) a R618L substitution in the SH2 domain, which prevents Stat interactions with phosphorylated tyrosine residues (SH2 mutant). HA-tagged WT Stat5a
and the two Stat5a mutant proteins were transiently expressed in NE1
cells and then immunoprecipitated from control and EGF-stimulated
cultures using HA-specific mAb. Probing of Western blots with
ErbB1-specific antibody showed that neither mutation disrupted Stat5a
association with ErbB1 (Fig.
4A, top panel).
However, inactivation of the SH2 domain prevented EGF-induced tyrosine
phosphorylation of Stat5a (Fig. 4A, middle
panel). Surprisingly, the Y694F mutant still displayed tyrosine
phosphorylation in response to EGF (Fig. 4A, middle
panel). To test whether this was specific to EGF, we transiently
expressed the Stat5a variants together with the prolactin receptor in
NE1 cells and analyzed prolactin-induced Stat5a tyrosine
phosphorylation. Prolactin is known to promote Stat5a activation via
Tyr694 phosphorylation (38). As seen with EGF, prolactin
failed to induce phosphorylation of the SH2 domain mutant (Fig.
4B). However, in striking contrast to EGF, the mutant
lacking Tyr694 was not phosphorylated in response to
prolactin (Fig. 4B). Apparently, EGF induces phosphorylation
of Tyr694 and additional tyrosine residues(s). The signal
is not because of dimerization and coimmunoprecipitation of endogenous
Stat5 molecules because EGF also induced tyrosine phosphorylation of the Y694F mutant in COS7 cells, which do not express Stat5 proteins (not shown). The lower panels in Fig. 4, A and
B show that equal amounts of Stat5a were expressed and
immunoprecipitated from all the cell lysates.
Next we tested the ability of the different Stat mutants to bind DNA by
performing an EMSA with the 32P-labeled Stat5 binding site
from the
The results indicate that the stable association of Stat5 with ErbB1 is
not mediated via interaction of its SH2 domain with a basally
phosphorylated receptor. However, the SH2 domain is apparently
necessary for ensuing EGF-induced tyrosine phosphorylation. The fact
that EGF, as opposed to prolactin, promotes Stat5a phosphorylation on
additional tyrosine(s) makes it tempting to speculate that the ErbB
receptor-mediated activation of Stat transcription factors occurs by an
alternate mode of signaling.
c-Src Is Activated by EGF and Recruited to the Stat-ErbB1
Complex--
The mechanism underlying ErbB receptor-mediated
activation of Stat transcription factors has not been resolved.
In vitro, ErbB1 is able to phosphorylate Stat1 and Stat3 (9,
10). However, it is not known whether additional kinases are involved
in vivo. In v-Src transformed cells, Stat3 is constitutively
activated and complexed with V-Src (15, 17), suggesting that this
kinase might be involved in Stat3 phosphorylation. Because c-Src lies on a pathway downstream of ErbB1 and ErbB2 (18-20), we examined its
role in ErbB receptor-induced phosphorylation of Stats. c-Src was
immunoprecipitated from EGF-treated A431 cells, and its activity was
measured in an in vitro kinase assay using acid-denatured enolase as a substrate. After 30 min of EGF treatment, c-Src activity increased, reaching its maximum (3.2-fold) at 60 min (Fig.
5A).
Next we examined whether c-Src was associated with ErbB1 and Stat
proteins. In A431 cells, ErbB1 and both p84 and p91 Stat1 could be
detected in immunoprecipitates of c-Src (Fig. 5B). A low
degree of association was observed in nonstimulated cells. However,
increased amounts of c-Src were recruited to ErbB1 and Stat1 within 5 min of EGF treatment, and the highest level of complex formation was
seen after 60 min of EGF treatment (Fig. 5B). A
ligand-induced increase in the complex of c-Src with ErbB1 and ErbB2
was also observed in EGF-treated NE1 cells and NDF-treated NE2/4 cells,
respectively (not shown). Following transient coexpression of HA-tagged
Stat5a and c-Src in A431 cells, association of these two proteins was
readily detected (not shown). The fact that the c-Src-ErbB1 interaction
peaked after 60 min of EGF stimulation, the time at which c-Src kinase
activity reached its maximum, supports the hypothesis that c-Src
creates its own binding site on ErbB1 (39).
Src Kinase Activity Is Required for ErbB Receptor-mediated Stat
Activation--
To explore the role of Src kinases in ErbB
receptor-mediated activation of Stat proteins, we examined the effect
of two nonrelated pharmacological Src kinase inhibitors, PP1 (27) and
CGP77675 (28), on EGF- and NDF-induced Stat phosphorylation. Prior to growth factor treatment, NE1 cells and NE2/4 cells were treated for 90 min with 10 µM PP1 or 1 µM CGP77675. Both
PP1 and CGP77675 completely blocked Stat5b tyrosine phosphorylation in
EGF-treated NE1 cells and NDF-treated NE2/4 cells (Fig.
6A). To determine whether Src
kinases were also involved in Stat1 and Stat3 activation, we
investigated the effects of the inhibitors in A431 cells. Both inhibitors abolished EGF-induced tyrosine phosphorylation of Stat1, Stat3, and Stat5b (Fig. 6B). Despite this block in Stat
activation, the inhibitors had little or no effect on ligand-induced
tyrosine phosphorylation of ErbB1, ErbB2, and ErbB4 (data not shown).
Furthermore, in NE1 cells, EGF-induced activation of extracellular
signal-regulated kinase 1/2 and p38 MAP kinases was not altered in the
presence of PP1 or CGP77675, indicating that receptor signaling was not impaired (data not shown).
In a second approach, we examined the effect of a DN c-Src protein on
Stat5a phosphorylation. HA-tagged Stat5a was coexpressed with either
the wild type or the DN mutant of c-Src in NE1 cells. Whereas
expression of wild type c-Src resulted in constitutive phosphorylation
of Stat5a, the level of which could not be increased any further by EGF
(Fig. 6C, lanes 3 and 4), expression
of DN c-Src inhibited both basal and EGF-induced tyrosine
phosphorylation of Stat5a (Fig. 6C, lanes 5 and
6).
Phosphorylation on the conserved C-terminal tyrosine residue in
Stats is essential for dimerization and specific DNA binding (4, 5).
Because Src inhibitors prevented ligand-induced Stat phosphorylation,
we also expected them to affect Stat DNA binding. To test this, total
cellular extracts from A431 cells treated with 5 nM EGF,
with or without preincubation with Src inhibitors, were prepared, and
an EMSA analysis was performed. Two bands were observed after EGF
treatment (Fig. 6D). Supershift analyses with specific
antibodies revealed that the more rapidly migrating band contained
Stat1 and the slower band contained Stat5 (not shown). Extracts from
inhibitor-treated cells displayed a marked reduction in DNA binding
activity (Fig. 6D). Tyrosine phosphorylation of Stat1 and
Stat5 was completely abolished by both Src inhibitors (Fig.
6B), yet DNA binding activity was not fully inhibited by the
PP1 inhibitor. This is most likely because of the higher amounts of EGF
used to visualize DNA binding (5 nM versus 1 nM for the experiment in Fig. 6B), whereas the
inhibitor concentration was not increased. Thus, inhibition of Src
kinase activity blocks both tyrosine phosphorylation and DNA binding of
Stat proteins. Taken together, these results support the idea that
functional Src kinases are required for ErbB receptor-mediated Stat signaling.
Jak1 and Jak2 Are Phosphorylated in Response to EGF and NDF and Are
Complexed with ErbB Receptors--
IFNs and cytokines activate Stat
transcription factors via Jak kinases (4, 5). However, the role of Jak
kinases in EGF-mediated Stat activation is not well understood. In an
attempt to establish Jak function in ErbB receptor signaling we
analyzed ligand-induced phosphorylation of these kinases in NIH
derivatives and in A431 cells.
Following EGF and NDF treatment of NE1 and NE2/4 cells, respectively,
an increase in Jak2 phosphorylation was observed (Fig. 7A, top panel). In
A431 cells, the ErbB1 agonists EGF, heparin-binding EGF, and
betacellulin but not NDF promoted Jak2 phosphorylation (Fig.
7B, top panel). This pattern of Jak2
phosphorylation correlates with Stat5 tyrosine phosphorylation in
response to these ligands (see Fig. 1C). Interestingly, a
ligand-induced tyrosine-phosphorylated band of approximately 180 kDa,
the size of ErbB receptors, coimmunoprecipitated with Jak2 (Fig. 7,
A and B, top panels, see
arrowheads). Reprobing of the membranes revealed that in
extracts from A431 cells, ErbB1 was present in Jak2 immunoprecipitates
(Fig. 7B). The sensitivity, however, was too low to allow
detection of coimmunoprecipitating ErbB1 in extracts from NE1 cells.
Similarly, ErbB2 could be detected in Jak2 immunoprecipitates from A431
and NE2/4 cells (Fig. 7, A and B, bottom
panels). Both receptors were associated with Jak2 even in
nonstimulated cells.
Next we examined the kinetics of Jak2 phosphorylation and its
association with ErbB1. Treatment of A431 cells with 1 nM
EGF led to a strong increase in Jak2 phosphorylation, which was rapid and sustained for 30 min (Fig.
8A). After EGF treatment, a
slight increase in the complex of the two proteins was noted, which
reached its maximum after 30 min. This was also observed when probing for associated Jak2 in ErbB1 immunoprecipitates (Fig.
8B).
Higher concentrations of EGF were necessary to visualize
phosphorylation of Jak1 in A431 cells. At a concentration of 16 nM (100 ng/ml), Jak1 phosphorylation was rapid and was
still elevated even after 2 h of growth factor treatment (Fig.
8C). The tyrosine-phosphorylated band of approximately 180 kDa at the top of the membrane is most likely ErbB1. Several additional
EGF-responsive, tyrosine-phosphorylated proteins coimmunoprecipitated
with Jak1 (see arrowheads), demonstrating that many proteins
physically interact with Jak1.
Jaks Are Downstream of Src but Are Not Required for ErbB
Receptor-induced Stat Activation--
Our results provide evidence
that Src kinases are required in ErbB receptor-mediated Stat signaling.
We have also shown that Jak kinases are constitutively associated with
ErbB receptors and become phosphorylated in response to ligand binding.
To examine the possibility that Src kinases mediate Stat activation via
Jaks, the effect of Src inhibitors on the phosphorylation of Jak2 was examined. The EGF-induced increase in Jak2 tyrosine phosphorylation was
completely abolished by pretreatment of A431 cells with either PP1 or
CGP77675 (Fig. 9A), suggesting
that Src kinases are upstream of Jaks. To control for specificity of
the inhibitors, we analyzed their effects on prolactin-induced
activation of Stat5, which is known to be mediated by Jak2 (38).
Treatment of HC11 mammary epithelial cells with the lactogenic hormones
dexamethasone, insulin, and prolactin leads to Stat5 activation (30).
Pretreatment of HC11 cells with PP1 or with CGP77675 had no effect on
dexamethasone, insulin, prolactin-induced Stat5 tyrosine
phosphorylation (Fig. 9B) or DNA binding activity (not
shown). Thus, the Src inhibitors do not have nonspecific effects on Jak
kinases and do not block Stat activation by lactogenic hormones.
Finally, we tested whether Jak activation was necessary for Stat
phosphorylation by performing transient transfection studies with DN
Jak constructs. HA-tagged Stat5a and DN Jak1 or DN Jak2 were
transiently expressed in NE1 cells. No inhibition of EGF-induced Stat5a
phosphorylation was observed (Fig. 10,
lanes 1-6). Coexpression of HA-tagged Stat5a, the prolactin
receptor, and DN Jak2 completely blocked prolactin-induced Stat5a
phosphorylation (Fig. 10, lanes 7-12), demonstrating that
DN Jak2 was capable of interfering with Jak2 function in our assay.
Finally, we conclude that, in ErbB receptor signaling, Src kinases
promote Stat1, Stat3, and Stat5a/b phosphorylation by a direct,
Jak-independent mechanism.
Stat transcription factors are activated by numerous cytokines and
peptide growth factors (4-6). EGF activates Stats in vitro in cultured cell lines (33, 35, 36, 40) and in vivo in the
liver (34). Our results show that in A431 cells, ErbB1 is constitutively associated with latent Stat1, Stat3, Stat5a/b, and Jak
kinases even in the absence of EGF. Ligand-induced recruitment of
active Src kinases to the receptor complex appears to be a prerequisite
for both Stat and Jak phosphorylation. Src kinases phosphorylate Stats,
in a Jak-independent fashion, on the conserved C-terminal tyrosine
residue, thereby promoting Stat dimerization, nuclear translocation,
and DNA binding. As opposed to prolactin, EGF induces Stat
phosphorylation on additional tyrosine(s), the function of which remain
to be elucidated. A model of the proposed mechanism by which ErbB
receptors mediate Stat activation is presented in Fig.
11.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 M dexamethasone, 5 µg/ml insulin, and
5 µg/ml ovine prolactin) (30).
-glycerophosphate, 2 mM sodium orthovanadate). Protein
extracts were obtained by three cycles of freezing and thawing followed
by clarification by centrifugation at 16,000 × g for
10 min. Nuclear extracts were prepared by scraping cells into CEB (10 mM KCl, 20 mM Hepes, pH 7.0, 1 mM
MgCl2, 0.1% Triton X-100, 20% glycerol, 0.1 mM EGTA, 0.5 mM dithiothreitol, 2 mM sodium orthovanadate, 50 µM
-glycerophosphate, 50 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin) and sheared with 20 strokes using a Dounce homogenizer (Wheaton, pestle B). Nuclei were pelleted by centrifugation at 800 × g for 5 min and then extracted with NEB (CEB + 300 mM NaCl) by incubating for 30 min on ice. Extracts
were clarified by centrifugation for 15 min at 16,000 × g. EMSAs were performed by incubating 10 µg of hypertonic
cell lysate or 10 µg of nuclear protein with the Stat5 DNA binding
site from the bovine
-casein promoter (5'-AGATTTCTAGGAATTCAATCC-3')
(31) for 30 min on ice in 20 µl of EMSA buffer (10 mM
Hepes, pH 7.6, 2 mM NaH2PO4, 0.25 mM EDTA, 1 mM dithiothreitol, 5 mM
MgCl2, 80 mM KCl, 2% glycerol, and 100 µg/ml
poly(dI-dC)). Specific binding was analyzed on a 4% native
polyacrylamide gel and prerun for 2 h at 200 V in 0.25 × TBE
(22.5 mM Tris borate, pH 8.0, 0.5 mM EDTA). The
samples were loaded and electrophoresed for 1 h at 200 V; the gels
were dried and autoradiographed. For supershift assays, nuclear
extracts were preincubated with antibody for 15 min prior to the
addition of the labeled probe.
-glycerophosphate, 15 mM sodium pyrophosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride) for 10 min on ice. The
lysates were clarified by centrifugation at 16,000 × g
for 10 min. For immunoprecipitations, equal amounts of protein were
incubated with specific antibodies for 2 h on ice. Immune
complexes were collected with protein A- or protein G-Sepharose
(Sigma), washed three times with lysis buffer, and washed once with TNE
buffer (50 mM Tris, pH 7.5, 140 mM NaCl, 5 mM EDTA). Precipitated proteins were released by boiling in
sample buffer and were subjected to sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE). The proteins were
blotted onto polyvinylidene difluoride membranes (Roche Molecular
Biochemicals). After blocking with 20% horse serum (Life Technologies,
Inc.) in TTBS (50 mM Tris, pH 7.5, 150 mM NaCl,
0.05% Tween 20), filters were probed with specific antibodies.
Proteins were visualized with peroxidase-coupled secondary antibody
using the ECL detection system (Amersham Pharmacia Biotech). Stripping
of membranes was performed in SDS buffer (62.5 mM Tris, pH
6.8, 2% SDS, 100 mM
-mercaptoethanol) for 30 min at
60 °C; membranes were then washed in TTBS and reprobed with the
indicated antibodies.
-32P]ATP. The reaction was stopped with sample
buffer; proteins were subjected to SDS-PAGE (10% gel) and blotted onto
polyvinylidene difluoride membranes. The membrane was analyzed using a
PhosphorImager (Molecular Dynamics) and ImageQuant software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ErbB receptor-mediated activation of
Stat5. NIH sublines (A and B) and A431
(C) cells were serum-starved for 18 h prior to
stimulation with growth factors at 1 nM for 10 min. Cells
were lysed, and Stat5b was immunoprecipitated with specific antiserum.
Immune complexes were subjected to SDS-PAGE and analyzed by Western
blotting with a phosphotyrosine-specific mAb. Membranes were stripped
and reprobed with Stat5b-specific antiserum. B, the origin
of the doublet present in the phosphotyrosine Western blot is unknown.
It could be a phosphotyrosine-containing protein that
coimmunoprecipitates with Stat5b, because reprobing with Stat5b
antiserum revealed a single band. BTC, betacellulin;
HB, heparin binding; PY, phosphotyrosine;
IP, immunoprecipitation; WB, Western blot.
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Fig. 2.
Time course of EGF-induced Stat
phosphorylation in A431 cells. A431 cells were serum-starved
for 18 h and, prior to lysis, were stimulated with 1 nM EGF for the indicated times. Stat1 (A), Stat3
(B), Stat5a (C), and Stat5b (D) were
immunoprecipitated with Stat-specific antibodies. Immune complexes were
resolved by SDS-PAGE and blotted. Membranes were probed with
phosphotyrosine-specific mAb and, after stripping, reprobed with the
respective Stat-specific antibodies. WB, Western blot;
PY, phosphotyrosine; IP,
immunoprecipitation.
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Fig. 3.
Stat1, Stat3, and Stat5 are associated with
ErbB1. A431 cells were serum-starved for 18 h and, prior to
lysis, were stimulated with 1 nM EGF for the indicated
times. C, HA-tagged Stat5a was transiently introduced into
A431 cells 8 h prior to serum starvation and subsequent growth
factor treatment. A and B, ErbB1 was
immunoprecipitated with mAbs EGFR1 and 528. C, Stat5a was
immunoprecipitated with HA-specific mAb. Immune complexes were resolved
by SDS-PAGE and blotted. A and B, the lower parts
of the membranes were probed with Stat1- and Stat3-specific antibodies,
respectively, and the top parts was probed with ErbB1-specific antibody
1005. C, The top part of the membrane was probed with
ErbB1-specific antibody 1005, and the lower part was probed with
HA-specific mAb. Lane on the far left, 50 µg of
WCE were loaded as a control. IP, immunoprecipitation;
WB, Western blot.
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Fig. 4.
Phosphorylation and activation of
Stat5a wild type and mutants by EGF and prolactin. A and
B, plasmids encoding HA-tagged WT Stat5a, Y694F Stat5a
(Y mut.), and R618L Stat5a (SH2 mut.) were
transfected into NE1 cells. B, a plasmid encoding the
prolactin receptor was cotransfected. 8 h posttransfection cells
were serum-starved for 18 h and, prior to lysis, were stimulated
with 1 nM EGF (A) for 10 min or 5 µg/ml
prolactin (PRL) (B) for 15 min. Stat5a was
immunoprecipitated with HA-specific mAb; immune complexes were resolved
by SDS-PAGE and blotted. The membrane was probed with
phosphotyrosine-specific mAb and, after stripping, reprobed with
Stat5a-specific antibody. A, the top part of the membrane
was probed with ErbB1-specific antibody 1005. C, plasmids
encoding HA-tagged WT, Y694F (Y mut.), and R618L (SH2
mut.) or an empty vector were transfected into NE1 cells. 8 h
posttransfection cells were serum-starved for 18 h and were
stimulated with 5 nM EGF for 15 min. Nuclear and
cytoplasmic extracts were prepared, and 10 µg of nuclear protein were
subjected to an EMSA using a radiolabeled Stat5 binding site as a
probe. Where indicated, samples were preincubated with 2 µg of
HA-specific mAb. D, 50 µg of cytoplasmic (C)
and nuclear (N) protein from the experiment described in
C were subjected to SDS-PAGE, blotted, and probed with
Stat5a-specific antiserum. PY, phosphotyrosine;
WB, Western blot.
-casein promoter (31). Nuclear extracts from EGF-treated
NE1 cells transiently expressing WT Stat5a demonstrated increased DNA
binding activity compared with extracts from cells transfected with
empty vector (Fig. 4C, lane 2 versus
11). This additional binding activity could be supershifted by HA-specific mAb (Fig. 4C, lane 3). The
remaining endogenous Stat binding activity could be supershifted with
Stat5-specific antiserum (not shown). The SH2 domain mutant had no
detectable DNA binding activity (Fig. 4C, lanes
7-9). Although still phosphorylated by EGF, the
Tyr694 mutant also failed to bind DNA (Fig. 4C,
lanes 4-6). Analysis of Stat5a present in cytoplasmic and
nuclear fractions showed that equal amounts of Stat5a wild type and
mutant proteins were expressed (Fig. 4D). Both mutants,
however, failed to translocate to the nucleus upon EGF stimulation
(Fig. 4D, lanes 8 and 12 versus lane 4). The low amount of Stat5a detected in these lanes is most likely because of translocation of endogenous Stat5a in response to
EGF.
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Fig. 5.
EGF-induced c-Src kinase activity and c-Src
association with ErbB1 and Stat1. A431 cells were serum-starved
for 18 h and, prior to lysis, were stimulated with 1 nM EGF for the indicated times. A, c-Src was
immunoprecipitated from 200 µg of WCE with mAb Ab-1, and immune
complexes were subjected to an in vitro kinase assay using
acid-denatured enolase as a substrate. Following SDS-PAGE and blotting,
c-Src activity was quantified using a PhosphorImager. The values
represent the mean of duplicate samples. B, c-Src was
immunoprecipitated with mAb Ab-1; immune complexes were resolved by
SDS-PAGE and blotted. The top part of the membrane was probed with
ErbB1-specific antibody 1005, and the lower part was probed with
Stat1-specific antibody and, after stripping, reprobed with
Src-specific mAb. B, 50 µg of WCE were loaded as a
control. WB, Western blot; IP,
immunoprecipitation.
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Fig. 6.
Src inhibitors and dominant negative c-Src
block ErbB receptor-mediated phosphorylation and DNA binding of
Stat proteins. A and B, NE1, NE2/4,
and A431 cells were serum-starved for 18 h and then treated with
10 µM PP1 or 1 µM CGP77675 for 90 min prior
to a 10 min stimulation with 1 nM EGF and NDF. Cells were
lysed, and Stat5b (A) and Stat1, Stat3, and Stat5b
(B) were immunoprecipitated with specific antibodies. Immune
complexes were resolved by SDS-PAGE and blotted. Membranes were probed
with phosphotyrosine-specific mAb and, after stripping, reprobed with
the respective Stat-specific antibodies. C, plasmids
encoding HA-tagged Stat5a and WT c-Src or DN c-Src were cotransfected
into NE1 cells. 8 h posttransfection, cells were serum-starved for
18 h and then stimulated with 1 nM EGF for 10 min
prior to lysis. Stat5a was immunoprecipitated with HA-specific mAb;
immune complexes were resolved by SDS-PAGE and blotted. The membrane
was probed with phosphotyrosine-specific mAb and, after stripping,
reprobed with HA-specific mAb. D, A431 cells were
serum-starved for 18 h and then treated with 10 µM
PP1 or 1 µM CGP77675 for 90 min prior to a 15 min
stimulation with 5 nM EGF. Proteins were extracted by
hypertonic lysis of cells and repeated cycles of freezing and thawing.
10 µg of protein were incubated with radiolabeled Stat5 binding site.
Complexes were electrophoresed on a 4% native polyacrylamide gel.
PY, phosphotyrosine; WB, Western blot;
IP, immunoprecipitation.
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Fig. 7.
Ligand-induced phosphorylation of Jak2 in
A431, NE1, and NE2/4 cells. NE1, NE2/4 (A), and A431
(B) cells were serum-starved for 18 h and, prior to
lysis, were treated with the indicated growth factors at 1 nM for 10 min. Jak2 was immunoprecipitated using polyclonal
antibody C20. Immune complexes were resolved by SDS-PAGE and blotted.
The membranes were probed with phosphotyrosine-specific mAb and, after
stripping, with ErbB1-specific antibody 1005 and ErbB2-specific serum
21N. The membranes were reprobed with Jak2-specific antiserum (UBI).
PY, phosphotyrosine; WB, Western blot;
IP, immunoprecipitation.
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Fig. 8.
Time course of Jak phosphorylation and
Jak/ErbB1 association. A431 cells were serum-starved for 18 h
and, prior to lysis, were stimulated with 1 nM EGF
(A and B) or 16 nM EGF (C)
for the indicated times. Jak2 was immunoprecipitated with polyclonal
antibody C20 (A), ErbB1 with antibody 1005 (B),
and Jak1 with specific polyclonal antiserum (C) (UBI).
Immune complexes were resolved by SDS-PAGE and blotted. A
and C, membranes were probed with phosphotyrosine-specific
mAb and, (A) after stripping, probed with ErbB1-specific
antibody 1005. (B, the membrane was probed with
Jak2-specific antiserum (UBI). All membranes were reprobed using
specific antibodies (A and B, not shown for
C). A and B, 50 µg of WCE were
loaded as a control. PY, phosphotyrosine; WB,
Western blot; IP, immunoprecipitation.
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Fig. 9.
Src inhibitors block EGF-induced
phosphorylation of Jak2 but do not inhibit lactogenic hormone-induced
activation of Stat5. A, A431 cells were serum-starved
for 18 h and then treated with 10 µM PP1 and 1 µM CGP77675 for 90 min prior to a 10 min stimulation with
1 nM EGF. Cells were lysed, and Jak2 was immunoprecipitated
with polyclonal antibody C20. Immune complexes were resolved by
SDS-PAGE and blotted. The membrane was probed with
phosphotyrosine-specific mAb and, after stripping, reprobed with
Jak2-specific antiserum (UBI). B, HC11 cells were
serum-starved for 12 h and then treated with 10 µM
PP1 and 2 µM CGP77675 for 90 min prior to a 15 min
stimulation with lactogenic hormones. Cells were lysed, and Stat5a was
immunoprecipitated with specific antiserum. Immune complexes were
resolved by SDS-PAGE and blotted. The membrane was probed with
phosphotyrosine-specific mAb and, after stripping, reprobed with
Stat5a-specific antiserum. DIP, dexamethasone, insulin,
prolactin; PY, phosphotyrosine; WB, Western blot;
IP, immunoprecipitation.
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Fig. 10.
Jak kinases are not required for EGF-induced
phosphorylation of Stat5. Plasmids encoding DN Jak1 or Jak2 were
cotransfected with a plasmid encoding HA-tagged Stat5a and, where
indicated, prolactin receptor (Prl-R) into NE1 cells. 8 h posttransfection cells were serum-starved for 18 h and, prior to
lysis, were stimulated with either 1 nM EGF for 10 min or 5 µg/ml prolactin for 15 min. Stat5a was immunoprecipitated with
HA-specific mAb; immune complexes were resolved by SDS-PAGE and
blotted. The membrane was probed with phosphotyrosine-specific mAb and,
after stripping, reprobed with HA-specific mAb. PY,
phosphotyrosine; WB, Western blot; IP,
immunoprecipitation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Model for ErbB receptor-mediated Stat
activation. EGF binds to its receptor, ErbB1, which is
preassociated with latent Jak and Stat proteins and induces receptor
dimerization, autophosphorylation, and recruitment of Src tyrosine
kinase. Src phosphorylates Jaks, and it phosphorylates Stats on the
consensus C-terminal tyrosine residue, which enables dimerization and
nuclear translocation, and on additional tyrosine(s). P,
phosphotyrosine.
Using NIH3T3 sublines expressing defined combinations of ErbB proteins (13), we examined the ability of distinct receptor dimers to induce Stat activation. In a previous study we have shown that ErbB2 can potentiate EGF-induced signaling through MAP and S6 kinases (14). However, ErbB2 expression did not enhance EGF-induced Stat activation, even when ErbB3 or ErbB4 were coexpressed. This suggests that ErbB1 homodimers are sufficient for maximal Stat activation. We provide the first report showing NDF induction of Stat activity, specifically through heterodimers of ErbB2 and ErbB4. We have recently shown that ErbB receptor phosphorylation is modulated by the dimerization partner (13). This explains how heterodimers can acquire novel signaling properties that are not the sum of the activity of the individual receptors. Intriguingly, tryptic phosphopeptide mapping of ErbB2 from NDF-treated NE2/4 cells revealed a phosphorylated peptide that was not present in ErbB2 from NDF-treated NE2/3 cells (13). It is tempting to speculate that this peptide (h) contains a phosphorylated residue involved in Stat activation by the ErbB2/ErbB4 heterodimer. The significance of heterodimer-specific signaling is seen in cardiac development where trabeculae formation absolutely depends on the presence of NDF and the coexpression of ErbB2 and ErbB4 (reviewed in Ref. 41). Therefore, in specific tissues NDF induction of the Stat pathway may be of biological importance.
NDF-activated ErbB2/ErbB3 dimers did not induce Stat phosphorylation, although we observed c-Src activation downstream of the ErbB2/ErbB3 dimer in SKBR3 cells (42). One striking difference between ErbB2/ErbB3 and ErbB2/ErbB4 dimers is their differential ability to activate the phosphatidylinositol 3-kinase (PI3K) pathway because of the multiple binding sites for the p85 subunit of PI3K on ErbB3 (43, 44). Treatment of cells with an inhibitor of PI3K, LY294002, led to increased phosphorylation of Stat5,2 suggesting that high levels of PI3K activity could negatively regulate Stat signaling. We are continuing to investigate the role of this pathway in Stat activation.
Our results show that there is constitutive association of Stat
proteins with ErbB1 in nonstimulated A431 cells. This is in contrast to
a previous report demonstrating ligand-induced recruitment of Stat1 to
ErbB1 (33). However, in these earlier experiments A431 cells were
serum-starved for 48 h, which may have disrupted basal Stat-ErbB1
complexes. There are other examples of Stat association with
nonstimulated receptors, e.g. Stat1 and Stat2 interact with a subunit of the IFN receptor in the absence of ligand (45). Fu and
Zhang (33) further reported that the SH2 domain of Stat1 was necessary
for association with ErbB1. Because we observed that the Stat5a SH2
domain mutant still complexed with ErbB1, Stat1 and Stat5a may interact
with the receptor via different mechanisms. The SH2 domain, however,
was necessary for ensuing EGF-induced tyrosine phosphorylation of both
Stat1 (33) and Stat5a (Fig. 4A).
Src family members have important roles in transducing mitogenic signals from receptor tyrosine kinases (reviewed in Refs. 21 and 22). In ErbB receptor signaling, we have identified a novel function of Src kinases as activators of Stat transcription factors. The pharmacological Src kinase inhibitors PP1 (27) and CGP77675 (28) used in this study blocked Stat activation in response to EGF and NDF. These inhibitors inactivate not only c-Src but also other Src family members. c-Src, c-Yes, and Fyn are ubiquitously expressed and thus, may act in concert to activate Stats. In addition to c-Src, c-Yes has been implicated in ErbB receptor signaling. Both kinases were found associated with ErbB2/Neu and demonstrated elevated kinase activity in Neu-induced tumors (20).
A431 cells have high c-Src kinase activity in the absence of growth factors (46), yet we detect no basal phosphorylation of Stat proteins. c-Src kinase activity peaked after 60 min of EGF treatment, whereas recruitment of c-Src to latent ErbB-Stat complexes was more rapid, occurring within 5 min of EGF treatment. Because Stat activation was also rapid, we consider ligand-induced recruitment of c-Src, rather than its level of activation, crucial for Stat and Jak phosphorylation. Interactions between Src and ErbB1 appear to be bidirectional. Src itself can phosphorylate ErbB1, leading to hyperactivation of the receptor and potentiation of downstream signals (47, 48). Because ErbB1 is able to phosphorylate Stat1 and Stat3 in vitro (9, 10), we cannot rule out the possibility that ErbB receptors hyperactivated by Src kinases directly phosphorylate Stats. c-Src is also involved in Stat3 activation in response to colony-stimulating factor 1 and interleukin-3 (15, 49). A more complex mechanism involving both Fyn and Jak2 activity has been proposed for Stat1 phosphorylation downstream of the angiotensin II receptor (50).
Tyrosine phosphorylation of Jak kinases in response to EGF (51) and platelet-derived growth factor (8) has been observed. However, the role of Jaks in receptor tyrosine kinase-mediated signaling is still unclear. Our results using DN Jak1 and Jak2 suggest that these kinases are not required for ErbB-induced Stat activation. This conclusion is supported by the fact that EGF- and platelet-derived growth factor-induced Stat activation were not affected in Jak1-, Jak2- and Tyk2-deficient cells (7, 8). This is the first report showing that ErbB receptor-induced Jak phosphorylation requires Src kinase activity. Elevated phosphotyrosine levels and increased activity of Jaks have been observed in v-Src transformed cells (52), suggesting that Jaks lie downstream of Src. However, this may be influenced by the cellular context because Jak phosphorylation was Src-independent in other studies (49, 50, 53).
We also present the first evidence of a direct interaction between ErbB
receptors and Jaks. Despite the fact that Jaks are apparently not
necessary for Stat activation, they may fulfill other important roles
in ErbB receptor signaling. Jaks can be phosphorylated on multiple
tyrosine residues, thereby providing docking sites for
phosphotyrosine-binding signaling proteins which, in turn, may serve as
substrates for Jak kinases. In addition to Stat activation, Jaks have
been implicated in the phosphorylation of Shc and the insulin-regulated
substrate 1/2 signaling molecules (54, 55) and in the activation of
Raf1 and the MAP kinase pathway in response to IFN, IFN
, and
oncostatin M (56, 57). Immunoprecipitates of Jak1 contained multiple
tyrosine-phosphorylated proteins (Fig. 8C), lending credence
to the hypothesis that Jaks have a function as scaffold molecules. Jaks
may in fact serve as adaptors for Stats, enabling interaction with ErbB
receptors. We show that Jaks are associated with ErbB1 and ErbB2.
Furthermore, we detected Jak2 in immunoprecipitates of transiently
expressed Stat5a (not shown). Indirect coupling of Stats to ErbB
receptors via constitutively associated Jaks is consistent with the
observation that autophosphorylation sites on ErbB1 are not required
for Stat activation (40).
In striking contrast to prolactin, EGF induced Stat5a phosphorylation
not only on the consensus Tyr694 but also on additional
tyrosine(s). We currently do not know whether this is specific for
Stat5a or applies to EGF-activated Stat proteins in general. In tryptic
phosphopeptide maps of EGF-activated Stat5a, we observed that
Tyr694 is the major site of
phosphorylation.3 Additional
phosphorylated tyrosine(s) may have a function in modulating Stat
activity by altering DNA binding or interaction with other
transcription factors. Alternatively, these tyrosine(s) may be involved
in signaling properties unrelated to transcriptional activation.
Because the Stat5a Tyr694 mutant failed to translocate to
the nucleus, it will be interesting to see if this mutant can
participate in other responses. For example, Stat3 has been proposed
to act as an adaptor protein, coupling PI3K to the IFN receptor via
phosphorylation on an additional tyrosine residue (58). Furthermore,
mutants of Stat1 proteins, which are deficient in dimer formation,
restored sensitivity to tumor necrosis factor-induced apoptosis in Stat
1/
cells (59).
Coexpression of ErbB1 and c-Src in murine fibroblasts results in a
synergistic increase in growth and tumorigenicity (47). Furthermore,
c-Src is activated/overexpressed in many human carcinomas that possess
elevated levels of ErbB receptors (60, 61). In this study we show that
Stats are downstream of both ErbB receptors and Src kinases. In NE1
cells, overexpression of Stat5a and wild type c-Src led to constitutive
activation of Stat5a (Fig. 6C), and in A431 cells, it led to
constitutive association of c-Src with Stat5a and ErbB1 (data not
shown). In addition, Src kinase inhibitors blocked the activation of
Stats by various ErbB ligands. Two recent reports demonstrated that
v-Src-induced malignant transformation is dependent on Stat3 (62, 63).
It is possible that Stat proteins are also involved in ErbB-mediated
tumorigenesis. However, the precise role of Stats in ErbB receptor
signaling awaits the elucidation of Stat target genes. It will be
interesting to determine whether Stats activated by Src kinases as
opposed to Jak kinases elicit different biologic responses.
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ACKNOWLEDGEMENTS |
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We thank M. El-Shemerly for helpful discussions, B. Groner for providing Prl-R expression plasmid, O. Silvennoinen for providing DN Jak1 and Jak2 plasmids, G. Superti-Furga for WT Src, S. Courtneidge for DN Src plasmids, J. Green for Src kinase inhibitor CGP77675, K. Ballmer-Hofer for mAb 327, Amgen for recombinant NDF, and H. Kaufmann for critical reading of the manuscript.
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FOOTNOTES |
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* 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.
Partially supported by a grant from the Schweizerische Krebsliga.
§ To whom correspondence should be addressed. Tel.: 41 61 697 8107; Fax: 41 61 697 8102; E-mail: hynes{at}fmi.ch.
2 M. A. Olayioye, unpublished data.
3 I. Beuvink, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; DN, dominant negative; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; IFN, interferon; Jak, Janus kinase; mAb, monoclonal antibody; MAP, mitogen-activated protein; NDF, neu differentiation factor; PI3K, phosphatidylinositol 3-kinase; SH2, Src homology 2; Stat, signal transducer and activator of transcription; WT, wild type; WCE, whole cell extracts; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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