(Received for publication, October 28, 1996, and in revised form, January 17, 1997)
From the Institute for Molecular and Cellular Biology, Signal transducers and activators of
transcription (Stat) proteins play an important role in signaling
through a variety of cytokine and growth factor receptors. Each of the
Stat proteins is activated in a ligand-specific manner. Only the Src
homology 2 (SH2) domains of Stat1 and Stat2 are critical for the
ligand-specific activation of interferon signaling. In this study we
determined the domains in Stat3 protein that contribute to interleukin
6 (IL-6)-specific phosphorylation. Based on evidence that Stat3, but
not Stat1, is activated in the presence of low levels of IL-6 and
soluble IL-6 receptor, we constructed various swap mutants between
Stat3 and Stat1 and examined their response to IL-6 after their
transient expression into COS7 cells. The region upstream of the SH2
domain was exchangeable between Stat1 and Stat3, whereas the region
carboxyl-terminal to the SH2 domain of Stat3 was critical to
phosphorylation by IL-6. However, unlike Stat1 and Stat2 in interferon
signaling, the swap mutant in which 5 amino acid residues just
carboxyl-terminal to the tyrosine phosphorylation site
(Tyr705) in Stat3 was replaced by the corresponding
region derived from Stat1 was not phosphorylated in response to IL-6.
Substituting 1 amino acid (Lys709) at position +4 relative
to Tyr705 abolished the tyrosine phosporylation of Stat3 in
response to IL-6. Co-immunoprecipitation experiments demonstrated that
these mutants were associated with gp130 at an extent similar to
wild-type Stat3. Taken together, these results show that the amino acid residues immediately carboxyl-terminal to the tyrosine phosphorylation site are involved in IL-6-specific activation of Stat3.
Cytokines and growth factors mediate their biological effects
through interaction with their receptors. Investigations into the
transcriptional response to interferons have identified the Janus
kinases (Jak)-signal transducers and activators of transcription (Stat)1 signaling pathway, which is in fact
used by a large number of cytokines and growth factors (1). The Jak
family of known nonreceptor protein kinases consists of Jak1, Jak2,
Jak3, and Tyk2. The family of Stat proteins constitutes a new class of
transcription factors that contains SH2, SH3-like domains, and a
carboxyl-terminal tyrosine phosphorylation site (2). Six Stat family
members (Stat1-Stat6) have been cloned. Jak kinases associate with the
cytoplasmic membrane proximal region of receptors and are catalytically
activated after ligand binding. The activated Jak kinases then
phosphorylate Stat proteins at their tyrosine residues. Thereafter, the
Stat proteins become homodimerized or heterodimerized and translocate
to the nucleus to activate transcription by interaction with specific DNA sequences.
The Stat proteins are activated in a ligand-specific manner. In
in vitro studies, Stat1 and Stat2 are phosphorylated in
response to IFN- The targeted disruption of the Stat genes in mice has revealed the
involvement of each Stat protein in the cytokine signal pathways
in vivo (22-27). Although Stat1 is activated by many
cytokines and growth factors, it appears specific for IFN pathways
in vivo. Stat1 To understand how the specificity of the signal is achieved after the
interaction of a ligand with the corresponding receptor and subsequent
activation of Jak kinases and Stat proteins, the molecular mechanism of
their interaction has been investigated. In response to IFN- We questioned whether the same is true of other members of the Stat
family. Here we performed structure-function analyses of Stat3 by
generating a series of swap mutants between Stat3 and Stat1 and
identified the region that contributes to the specificity of
IL-6-dependent Stat3 tyrosine phosphorylation. We confirmed the importance of the SH2 domain in Stat3 and showed that, besides the
SH2 domain, the specific activation of Stat3 by IL-6 is determined by
the amino acid sequences present just carboxyl-terminal to the tyrosine
phosphorylation site.
Recombinant human IL-6 and soluble
IL-6 receptor were provided by K. Yasukawa (Toso, Kanagawa, Japan).
Recombinant human EGF was purchased from Genzyme (Cambridge, MA). The
antibodies to Flag epitope and to phosphotyrosine were purchased from
IBI (New Haven, CT) and Upstate Biotechnology (Lake Placid, NY),
respectively.
COS cells were grown on plastic
culture dishes in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum. Myeloid leukemia M1 cells were cultured in Eagle's
minimal essential medium supplemented with twice the normal
concentrations of amino acids and vitamins and 10% fetal calf
serum.
Some constructs were generated by
introducing new restriction enzyme sites, which did not alter the amino
acid residue, by the polymerase chain reaction or by site-directed
mutagenesis (Transformer site-directed mutagenesis kit, Clontech).
Other constructs were directly produced by site-directed mutagenesis.
The subcloned polymerase chain reaction products were completely
sequenced. The constructs were tagged with the Flag epitope (Eastman
Kodak Co.) at their NH2 termini and cloned into the
mammalian expression vector pEF-BOS (35). The amino acid (aa)
boundaries of the chimeric proteins were as follows: 3/1A has aa 1-235
of Stat1 and 240-771 of Stat3; 3/1B has aa 1-325 of Stat1 and
330-771 of Stat3; 3/1C has aa 1-443 of Stat1 and 450-771 of Stat3;
3/1D has aa 1-596 of Stat1 and 604-771 of Stat3; 3/1E has aa 326-751
of Stat1 and 1-329 of Stat3; 3/1F has aa 449-751 of Stat1 and 1-453
of Stat3; 3/1G has aa 694-751 of Stat1 and 1-698 of Stat3; 3/1H has
aa 701-751 of Stat1 and 1-704 of Stat3; 3/1J has aa 705-715 of Stat3
substituted for 701-711 of Stat1; 3/1K has aa 705-710 of Stat3
substituted for 701-706 of Stat1; and 3/1L has aa 711-715 of Stat3
substituted for 707-711 of Stat1. Y705F was generated by site-directed
mutagenesis converting the tyrosine 705 of Stat3 to phenylalanine, and
K709E was generated converting lysine 709 of Stat3 to glutamate.
COS cells were transfected using the
DEAE-dextran method and cultured for 48 h in complete media before
being deprived of serum for 5 h. The cells were then incubated for
15 min with cytokines or growth factors, and then the cells were lysed
for immunoprecipitation. M1 cells were transfected via electroporation
with the expression vector and pSV2neo at a 20:1 ratio, and
neomycin-resistant clones were selected in growth medium containing 750 mg/ml of Geneticin (Life Technologies, Inc.).
Cells were
solubilized with Nonidet P-40 lysis buffer (1.0% Nonidet P-40 and 10 mM Tris HCl, pH 7.6, containing 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 5 mg/ml aprotinin,
leupeptin, and pepstatin) and then centrifuged. The clarified lysates
were incubated with 10 µg of M2 anti-Flag monoclonal antibody and
protein A-Sepharose (Pharmacia Biotech Inc.), and then
immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis and immunoblotted with M2 or anti-phosphotyrosine monoclonal antibody. Blots were visualized using the ECL detection system (Amersham Corp.) according to the manufacturer's
procedures.
We assessed the dose of IL-6 required to phosphorylate Stat1 and
Stat3. To discriminate exogenous and endogenous Stats, we constructed
expression vectors for Stat1 and Stat3, both of which were Flag
epitope-tagged on their amino termini. These vectors were transiently
transfected into COS cells and stimulated with increasing
concentrations of IL-6 together with soluble IL-6 receptor. The cells
were then lysed, immunoprecipitated with anti-Flag antibody, and
immunoblotted with anti-phosphotyrosine antibody. Fig.
1A shows that at an IL-6 concentration of 25 ng/ml, Stat3 was already fully phosphorylated, whereas Stat1 was not at
all or only weakly phosphorylated. As the concentration of IL-6
increased, Stat1 phosphorylation was augmented in a
dose-dependent manner, although the amount of
phosphorylation at 300 ng/ml IL-6 was still significantly lower than
that of Stat3 at 25 ng/ml. In contrast, Stat3 was maximally phosphorylated at 25 ng/ml IL-6. These results demonstrated that only
Stat3 is activated at a low concentration of IL-6, although Stat1 is
activated at a very high concentration. Therefore, we used 25 ng/ml
IL-6 to stimulate COS cells in the following experiments. We also
examined the tyrosine phosphorylation of wild-type Stat3 and Stat1 in
response to EGF. Both Stat3 and Stat1 were phosphorylated in COS cells
exposed to EGF (Fig. 1B).
To determine which domains contribute to the IL-6-specific activation
of Stat3 protein, we constructed several swap mutants at conserved
amino acids between Stat3 and Stat1, termed 3/1A-3/1D (Fig.
2). In construct 3/1A, the amino-terminal region of
Stat3 was substituted by the comparable region of Stat1. The constructs 3/1B and 3/1C contained Stat1 residues in place of Stat3 aa 1-329 and
1-449, respectively. In the construct 3/1D, the substituted region at
aa 1-596 encompassed the region from the amino terminus to part of the
SH2 domain. These constructs were transiently transfected into COS
cells stimulated with IL-6 and soluble IL-6 receptor or EGF. Then we
examined phosphorylation. Fig. 3 shows that IL-6 and EGF
activated all the constructs in which the region from the
amino-terminal to the upper half of the SH2 domain was substituted by
the corresponding region of Stat1. These results showed that the
amino-terminal region upstream of the SH2 domain is exchangeable between Stat1 and Stat3.
To examine the role of SH2 domains in the ligand-specific
phosphorylation of Stat3 and Stat1, we constructed swap mutants in
which the carboxyl-terminal regions of Stat3 were replaced by the
comparable regions of Stat1 (Fig. 4). In the construct 3/1E, the carboxyl-terminal region containing the DNA binding domain
(aa 330-771) of Stat3 was substituted by that of Stat1. In the
construct 3/1F, the region containing the SH3 and downstream domains
(aa 454-771) was replaced by that of Stat1. Fig. 5
shows that IL-6 did not activate the 3/1E and 3/1F mutants. In
contrast, EGF phosphorylated both of them. Together with the results
shown in Fig. 3, these data indicated that the carboxyl-terminal region containing the SH2 and its downstream domains provide the specificity for Stat3 phosphorylation mediated by IL-6 but not by EGF.
We and other groups have demonstrated that the SH2 domain is critical
for Stat3 phosphorylation because a single amino acid substitution of
arginine to glutamine in the SH2 domain completely abolished Stat3
phosphorylation. We examined whether only the SH2 domain is sufficient
for IL-6-specific phosphorylation. We constructed the swap mutant,
3/1G, in which the region carboxyl-terminal to the SH2 domain (aa
699-771) was replaced by that of Stat1 (Fig. 4). IL-6 did not
phosphorylate this mutant (Fig. 5). Even when the replaced region (aa
705-771) was narrowed at the tyrosine residue to be phosphorylated,
Tyr705 (this mutant is named 3/1H), IL-6 did not activate
the mutant. However, when the swap site was placed 5 amino acids
carboxyl-terminal to Tyr705 (aa 710), the mutant was
phosphorylated. These data suggested that the carboxyl-terminal
boundary of the critical region for IL-6-specific Stat3 phosphorylation
lies within the 5 amino acid residues carboxyl-terminal to
Tyr705.
To determine the amino acid residues critical for the IL-6-specific
phosphorylation of Stat3, we constructed the swap mutants 3/1J, 3/1K,
and 3/1L (Fig. 6A). The corresponding Stat1
residues were substituted for the tyrosine phosphorylation site and the downstream 10 amino acid residues (aa 705-715) of Stat3 in 3/1J. The 5 amino acids critical for the IL-6-specific phosphorylation of Stat3 (aa
706-710) were replaced by the corresponding region of Stat1 in 3/1K.
Five downstream amino acids (aa 711-715) adjacent to the critical 5 amino acids were replaced by the corresponding region of Stat1 in 3/1L.
Fig. 6B shows that EGF and IL-6 phosphorylated 3/1L, whereas
only EGF phosphorylated 3/1J and 3/1K. The amino acid difference
between the 5 amino acids critical for IL-6-specific phosphorylation of
Stat3 and the corresponding 5 amino acids of Stat1 consists of 3 residues: +1 (Leu
To exclude the possibility that these results were specific to COS
cells, we generated permanent M1 clones that harbor wild-type Stat3 and
several swap mutant expression vectors. M1 is a mouse myeloid leukemia
cell line that differentiates into macrophages in response to IL-6. M1
cells did not respond to EGF, but wild-type Stat3 and 3/1L were
phosphorylated by stimulation with IL-6 and soluble IL-6 receptor
whereas, 3/K was not (Fig. 7). As in COS cells, the 5 amino acid residues carboxyl-terminal to Tyr705 were
critical for the IL-6-specific phosphorylation of Stat3 in M1
cells.
To examine the reason why IL-6 did not tyrosine phosphorylate the
carboxyl-terminal swap mutants in response to IL-6, we studied their
association with gp130. COS cells were co-transfected with the
expression vectors for wild-type Stat3 or its mutants and a gp130
expression vector. After stimulation with IL-6 and soluble IL-6
receptor, the lysates were immunoprecipitated with anti-Flag antibody
and then blotted with anti-phosphotyrosine and anti-Flag antibodies,
respectively (Fig. 8). Anti-Flag antibody
co-precipitated gp130 in COS cells transfected with the vector
expressing wild-type Stat3, indicating that wild-type Stat3 associates
with gp130. In COS cells transfected with the vector expressing
wild-type Stat1 and stimulated with IL-6 and soluble IL-6 receptor,
Stat1 was neither phosphorylated nor associated with gp130.
In contrast, all the swap mutants, 3/1J, 3/1K, 3/1L, and K709E,
co-immunoprecipitated gp130, although only 3/1L was phosphorylated, as
shown in Fig. 6B (Fig. 8). These results indicate that the 5 amino acid residues carboxyl-terminal to Tyr705 that were
critical for the IL-6-specific phosphorylation did not play a role in
the association with gp130.
There is some discrepancy among studies of Stat protein activation
by IL-6 treatment. Some have shown that only Stat3 is activated (11,
13), whereas others have found that both Stat3 and Stat1 are activated
(36, 37). In addition to IFNs, Stat1 is tyrosine-phosphorylated by many
factors, such as EGF, platelet-derived growth factor, LIF, IL-6, IL-10,
colony-stimulating factor 1, and angiotensin. However, the doses of
these factors were not considered in these studies. In this study, Stat
transient expression in COS cells and immunoblotting showed that only
Stat3 is phosphorylated at low concentrations of IL-6, whereas high
concentrations activated both Stat3 and Stat1. The
dose-dependent selectivity of Stat activation has been
shown by means of an electrophoretic mobility shift assay (6). In HepG2
cells at a low IL-6 concentration, SIF-A (a Stat3 homodimer) was the
predominant DNA binding complex induced, whereas at higher IL-6
concentrations, SIF-B (a Stat3 and Stat1 heterodimer) and SIF-C (a
Stat1 homodimer) were also formed. Although Stat1 is activated by IL-6,
Stat3 plays a critical role in the biological effects exerted by IL-6.
In myeloid leukemia M1 cells that undergo terminal differentiation and
growth arrest in response to IL-6, overexpression of the dominant
negative forms of Stat3 prevented both growth arrest and terminal
differentiation (38, 39). On the contrary, despite its activation by
factors other than IFNs, Stat1 is essential for IFN signaling, because
Stat1-deficient mice display a complete lack of responsiveness to
either IFN- The amino-terminal region is conserved in Stat proteins and has
multiple functions. The amino terminus of Stat1 is important in
modulating its phosphorylation, possibly by interacting with a
phosphatase (43). The domain also contributes to the co-operative DNA
binding and enables the Stat proteins to recognize variations of the
consensus site (44). Deletion mutants of Stat1 lacking the
amino-terminal conserved region were not phosphorylated by IFN- In this study, we showed that the carboxyl-terminal region, including
the SH2 domain, determines the specificity between Stat3 and Stat1.
Heim et al. (34) have also demonstrated that the SH2 domain
plays a critical role in the selective activation of Stat1 and Stat2 in
response to IFNs. Changing the Stat1 SH2 domain to that of Stat2
prevented IFN- Heim et al. (34) demonstrated in the same study that the
Stat1 chimeric constructs harboring the Stat2 sequence surrounding the
tyrosine phosphorylation site can be normally activated in response to
IFN- All swap mutants between Stat1 and Stat3 were equally associated with
the EGF receptor and phosphorylated. Stat1 and Stat3 are both activated
through the EGF receptor (4, 6, 8, 50). Studies using the EGF receptor
deletion mutants showed that two intracellular tyrosine residues
(Tyr1068 and Tyr1086) are necessary for Stat
activation (51) and may provide binding sites for Stats. Although EGF
stimulates Jak phosphorylation on tyrosine residues (52), whether the
receptor activates Stats directly or through Jaks is in dispute.
However a study using Jak1-defective cells has revealed that the kinase
activity of the receptor but not Jak1 is critical for Stat activation
(53). Thus in EGF both the mechanism of recruitment and phosphorylation might be different from gp130. It remains to be established whether the
amino acid sequence surrounding the tyrosine phosphorylation site is
required for the activation of other Stats in other cytokine and growth
factor signaling.
We thank K. Nakashima, M. Narazaki, and T. Taga for helpful discussion. We also thank K. Kubota for excellent
secretarial assistance.
Department of Internal Medicine III,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, whereas Stat1, but not Stat2, is phosphorylated in
response to IFN-
. Besides the IFN signaling systems, Stat1 is
activated by signaling through various cytokine receptors (3-6),
growth factor receptors (6-8), and the G protein-coupled receptor for angiotensin II (9, 10). Stat3 is phosphorylated by stimulation with
cytokines using gp130 and gp130-related receptors such as IL-6,
leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic
factor, and granulocyte colony-stimulating factor, as well as EGF
(11-13). Stat4 and Stat6 are phosphorylated in response to IL-12 and
IL-4, respectively (14, 15). Stat5 is phosphorylated by a variety of
cytokines, including IL-2, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, prolactin, and thrombopoietin (16-21).
/
embryonic stem cells are unresponsive to
IFN but retain responsiveness to LIF and remain
LIF-dependent for undifferentiated growth (22), indicating
that Stat1 does not play a distinctive role in LIF signaling. Stat6
plays an essential and specific role in IL-4 (24, 25), and Stat4 does
in IL-12 signaling (26, 27).
, Stat1
is activated by Jak1 and Jak2. A functionally critical membrane-distal
tyrosine residue (Tyr440) in the IFN-
receptor is a
target of activated Jak kinases, and the sequence YpDKPH, containing
the phosphotyrosine of the IFN-
receptor, provides the specific
association site for Stat1 (28). In IFN-
signaling, Leung et
al. showed that Stat1 and Stat2 proteins might be sequentially
phosphorylated in response to IFN-
, and that phosphorylated Stat2
might be required, so that unphosphorylated Stat1 can bind to the
activated IFN-
receptor (29). For IL-6 signaling, gp130 becomes
homodimerized when IL-6 binds the IL-6 receptor (30, 31). Dimerization
induces intermolecular phosphorylation and activation of the associated
Jaks (Jak1, Jak2, and Tyk2), which then phosphorylate tyrosines on
gp130 (32). The box3 region of gp130 contains YXXQ, motifs
and it acts as a docking site that selectively binds Stat3, which is in
turn phosphorylated by activated Jaks (33). Using chimeric constructs between Stat1 and Stat2, Heim et al. (34) have shown that
the SH2 domain in Stat proteins plays a critical role in their
ligand-specific phosphorylation of Stat proteins (34).
Reagents and Antibodies
Fig. 1.
A, dose-dependent
phosphorylation of Stat1 and Stat3 in response to IL-6. COS-7 cells
transfected with wild-type Stat3 or Stat1 were incubated for 15 min at
37 °C at the indicated concentrations of recombinant human IL-6
together with the soluble IL-6 receptor. Cells were lysed, and the
transfected Stats were immunoprecipitated with anti-Flag antibody M2.
Immunoprecipitates were Western blotted using the monoclonal
anti-phosphotyrosine antibody 4G10 and M2. Arrowhead,
position of transfected Stats; Ig, mouse immunoglobulin. B, phosphorylation of Stat1 and Stat3 in response to EGF.
COS-7 cells were transfected with wild-type Stat3 or Stat1 and
stimulated with 50 ng/ml recombinant human EGF for 15 min. Cell lysates
were immunoprecipitated with the anti-Flag antibody M2.
Immunoprecipitates were Western blotted using the monoclonal
anti-phosphotyrosine antibody 4G10.
[View Larger Version of this Image (51K GIF file)]
Fig. 2.
Schematic representation of chimeric Stat
proteins between Stat1 and Stat3. Leucine repeat, DNA binding,
SH3, and SH2, subdomains conserved among members of the
Stat family; Y, tyrosine residue that is phosphorylated on
ligand stimulation. Open bars, wild-type Stat3 and the parts
originated from Stat3 in the chimeric proteins; shaded bars,
Stat1. Numbers on the boundaries, marginal amino acid
residues.
[View Larger Version of this Image (12K GIF file)]
Fig. 3.
The region upstream of the SH2 domain is
exchangeable between Stat1 and Stat3. COS-7 cells were transfected
with wild-type Stat3, Stat1, and the chimeric Stat3/1 proteins
described in Fig. 2. Serum-deprived cells were incubated for 15 min at
37 °C with or without 25 ng/ml recombinant human IL-6 and 25 ng/ml
soluble recombinant human IL-6 receptor. Cells were lysed and
immunoprecipitated with the anti-Flag antibody M2. Immunoprecipitates
were Western blotted using the monoclonal anti-phosphotyrosine antibody
4G10 (top panel) or the anti-Flag antibody M2 (bottom
panel).
[View Larger Version of this Image (55K GIF file)]
Fig. 4.
Schematic representation of chimeric Stat
proteins in which the carboxyl-terminal regions of Stat3 were
substituted by those of Stat1. Open bars, parts originated
from Stat3; shaded bars, Stat1. The abbreviations and
numbers are the same as those described in the legend to Fig. 2.
[View Larger Version of this Image (13K GIF file)]
Fig. 5.
The region carboxyl-terminal to the SH2
domain of Stat3 is important for phosphorylation by IL-6 but not by
EGF. COS-7 cells were transfected with the chimeric Stat3/1
proteins described in Fig. 4. Cells were stimulated without () or
with recombinant human IL-6 and soluble IL-6 receptor (6) or
recombinant human EGF (E) as described in the legend to Fig.
3. Cells were lysed and immunoprecipitated with the anti-Flag antibody
M2. Immunoprecipitates were Western blotted using the monoclonal
anti-phosphotyrosine antibody 4G10 (top panel) or the
anti-Flag antibody M2 (bottom panel).
[View Larger Version of this Image (39K GIF file)]
Ile), +4 (Lys
Glu), and +5 (Phe
Leu).
Since the substitution at +4 seemed to cause the biggest difference, we
changed +4 lysine to glutamate in Stat3 by site-directed mutagenesis
(mutant K709E). EGF, but not IL-6, phosphorylated this mutant.
Therefore, the +4 lysine is necessary for the IL-6-specific activation
of Stat3.
Fig. 6.
The amino acid residues downstream of the
tyrosine phosphorylation site contribute to IL-6-specific
phosphorylation of Stat3. A, schematic representation of
chimeric Stat3/1 proteins in which 10 amino acid residues downstream of
the tyrosine phosphorylation site (3/1J), the upstream 5 of
the 10 amino acids (3/1K), and the downstream 5 of the 10 (3/1L) were substituted by the corresponding regions of
Stat1. In mutant K709F, lysine 709 was changed to glutamine on Stat3.
Numbers of the marginal amino acids of Stat3 are placed on
the bars, and those of Stat1 are placed below. B, COS-7
cells were transfected with the chimeric Stat3/1 proteins described in
A. Cells were stimulated without () or with recombinant
IL-6 with soluble IL-6 receptor (6) or recombinant human EGF
(E) as described in the legend to Fig. 3. Cells were lysed
and immunoprecipitated with the anti-Flag antibody M2.
Immunoprecipitates were Western blotted using the monoclonal
anti-phosphotyrosine antibody 4G10 (top panel) or anti-Flag
antibody M2 (bottom panel).
[View Larger Version of this Image (22K GIF file)]
Fig. 7.
The 5 amino acid residues downstream of the
tyrosine phosphorylation site are critical to IL-6-specific
phosphorylation of Stat3 in M1 cells. M1 cells were transfected
via electroporation with vectors expressing wild-type Stat3 and the
chimeric Stat3/1 proteins 3/1K and 3/1L shown in Fig. 6 with pSV2neo.
Neomycin-resistant clones were selected. Cells were stimulated without
() or with recombinant human IL-6 and soluble IL-6 receptor
(6) or recombinant human EGF (E) as described in
the legend to Fig. 3. Cells were lysed and immunoprecipitated with
anti-Flag antibody M2. Immunoprecipitates were Western blotted using
the monoclonal anti-phosphotyrosine antibody 4G10 (top
panel) or anti-Flag antibody M2 (bottom panel).
[View Larger Version of this Image (59K GIF file)]
Fig. 8.
The 5 amino acid residues downstream of the
tyrosine phosphorylation site are not involved in Stat3
association. COS-7 cells were transfected with wild-type Stat3,
Stat1, 3/1J, 3/1K, 3/1L, or K709E together with a gp130 expression
vector. Cells were stimulated without () or with recombinant human
IL-6 and soluble IL-6 receptor (6) as described in the
legend to Fig. 3. Cells were lysed and immunoprecipitated with
anti-Flag antibody M2. Immunoprecipitates were Western blotted using
the monoclonal anti-phosphotyrosine antibody 4G10 (top
panel) or the anti-Flag antibody M2 (bottom
panel).
[View Larger Version of this Image (37K GIF file)]
or IFN-
but respond normally to other cytokines that
activate Stat1 in vitro (22, 23). To date, how Stat1 is
activated at high concentrations of IL-6 and what the function of Stat1
is in IL-6 signaling remain to be clarified. We and other investigators showed that gp130 is immunoprecipitated by activated Stat3 but not by
Stat1, even when phosphorylated at high concentrations of IL-6 (data
not shown). Stat1 may be activated by forming a heterodimer with Stat3
through docking to Stat3 phosphotyrosine, as demonstrated by the
activation of Stat1 in IFN-
signaling (29). Alternatively, Stat1 may
associate with gp130 in such a degree that recruitment cannot be
detectable by immunoprecipitation assay (40) or may directly associate
with activated Jak kinases and become phosphorylated. In fact, Gupta
et al. (41) have reported that the Stat1 SH2 domain mediates
an obligate interaction with the Jak kinases, indicating that
overstimulation causes the direct binding of Stat1 with Jak protein
kinases and subsequent phosphorylation by Jaks. Furthermore, very high
erythropoietin levels activate Stat5 even when an erythropoietin
receptor mutant lacks all intracellular tyrosines (42).
or
IFN-
, and the helical repeat within their amino-terminal regions is
necessary for Stat1 phosphorylation (45, 46). Our findings that these
regions are exchangeable between Stat3 and Stat1 suggested that the
amino-terminal region does not function as a specificity determinant in
their phosphorylation.
activation, whereas changing the SH2 domain of Stat2
to that of Stat1 allowed phosphorylation by IFN-
. It is most
probable that the exchange of the SH2 domains altered the specific
association of Stat proteins with the receptor complexes. In fact, the
SH2 domain of each Stat protein interacts with a specific amino acid
sequence containing phosphotyrosines in the receptors. The consensus
sequence YXXQ in the cytoplasmic domain of gp130 is the
critical determinant by which Stat3 is activated (33). The consensus
sequence (Y440DKP) of the IFN-
-chain may provide a binding site
for Stat1 (28). Stat6 binds two tyrosine phosphopeptides (GYPKAFS and
GYPKPFQ) derived from the intracellular domain of the IL-4 receptor
-chain.
or IFN-
. Therefore, they concluded that swapping the
tyrosine phosphorylation site and its surrounding amino acids between
Stat1 and Stat2 did not change the specificity of activation by IFN-
and IFN-
. However, this study showed that the amino acid sequence
carboxyl-terminal to Tyr705 in Stat3 contributes to
IL-6-specific Stat3 activation. The chimeric protein in which the amino
acid sequence immediately carboxyl-terminal to Tyr705 in
Stat3 was replaced by the corresponding sequence derived from Stat1 was
associated with gp130 but not activated, indicating that the SH2 domain
is sufficient for Stat3 to bind gp130 but some additional factors are
required for the phosphorylation of Stat3. When overexpressed, Jaks
equally activate all Stat proteins without ligand stimulation, so the
Jak kinase has been regarded as having no specificity for substrate
between Stat families (47, 48). We also confirm that our recombinant
Stat3/1 proteins were all activated by co-expressed Jak1 (data not
shown). In the case of receptor-mediated phosphorylation, some
topological constraints in the receptor-Jak-Stat complex may hinder the
access of Stat3/1 to the catalytic domain of Jak kinase. Kirshnan
et al. (49) also showed that there might be some favorable
geometry of the receptor-Jak-Stat complex to allow Jaks to
phosphorylate Stat2 in IFN-
signaling.
*
This work was supported by grants from the Ministry of
Education of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Biochemistry,
Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan.
1
The abbreviations used are: Stat, signal
transducers and activators of transcription; Jak, Janus kinase; SH, Src
homology; IFN, interferon; IL, interleukin; LIF, leukemia inhibitory
factor; EGF, epidermal growth factor; aa, amino acid(s).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.