From the Department of Biochemistry,
Rheinisch-Westfälische Technische Hochschule Aachen,
Pauwelsstrasse 30, Aachen D-52074, Germany, the ¶ Bayer Pharma
Research Center, Aprather Weg 18a, Wuppertal D-42096, Germany, the
Cancer Biology Program, Beth Israel Deaconess Medical Center,
Boston, Massachusetts 02215, the ** Molecular and
Cellular Immunology, Medical Institute of Bioregulation, Kyushu
University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan, and
the
Department of Medical Immunology,
Universitätsklinikum Charité, Molecular Libraries and
Recognition, Ziegelerstrasse 5-9, Berlin D-10117, Germany
Received for publication, October 15, 2002
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ABSTRACT |
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Interleukin-6 (IL-6) activates the Jak/STAT
pathway as well as the mitogen-activated protein kinase cascade.
Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has
been identified as being involved in negative regulation of
IL-6-induced gene induction and activation of the Jak/STAT pathway.
Because this site is known to be a recruitment motif for the
protein-tyrosine phosphatase SHP2, it has been suggested that
SHP2 is the mediator of tyrosine 759-dependent signal
attenuation. We recently observed that the suppressor of
cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of
gp130. However, the relative contributions of SHP2 and SOCS3 to the
repression of IL-6 signaling are not understood. Therefore, we designed
experiments allowing the independent recruitment of each of these
proteins to the IL-6-receptor complex. We show that receptor-
and membrane-targeted SHP2 counteracts IL-6 signaling independent of
SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in
cells expressing a truncated SHP2 protein, which is not recruited to
gp130. These data suggest, that there are two, largely distinct modes
of negative regulation of gp130 activity, despite the fact that both
SOCS3 and SHP2 are recruited to the same site within gp130.
Interleukin-6 (IL-6)1 is
a cytokine with a wide spectrum of activities. It stimulates the
differentiation of B- and T-cells cells and the proliferation of
keratinocytes, mesangial and plasmacytoma cells, whereas the
proliferation of breast carcinoma cells, as well as melanoma cells is
inhibited (for review see Ref. 1). IL-6 is the major regulator of the
expression of acute-phase protein genes in liver cells (2-5) and
exerts its action by binding and activating a receptor complex composed
of a specific Tyrosine 759 of gp130 has been suggested to be involved in the
inhibition of IL-6 signaling: mutation of tyrosine 759 to phenylalanine was shown to enhance signal transduction of IL-6 (14-16) as
well as signaling of leukemia inhibitory factor and oncostatin M (17). The latter cytokines signal through LIF-R/gp130 or OSM-R/gp130 heterodimeric receptor complexes, respectively. Mice expressing gp130,
which lack the tyrosine 759, display splenomegaly, lymphadenopathy, and
an enhanced acute phase reaction (18). On the other hand, mice
expressing C-terminally deleted gp130 lacking the STAT-binding sites
show a similar phenotype as IL-6-deficient mice (impaired humoral and
mucosal immune and hepatic acute phase response) (19).
The protein-tyrosine phosphatase SHP2 is believed to mediate
Tyr-759-dependent signal attenuation, because SHP2 is
recruited to phosphotyrosine 759 after receptor activation and becomes
tyrosine-phosphorylated (14-16). Mutation of Tyr-759 impairs SHP2
recruitment and phosphorylation (10). Furthermore, IL-6 induced
activation of the MAPK cascade is blocked by this receptor mutation.
SHP2 is a ubiquitously expressed cytoplasmic protein-tyrosine
phosphatase containing two N-terminal Src homology 2 (SH2) domains and
a phosphatase domain in the C-terminal half. The crystal structure of
SHP2 suggests that, in the absence of a tyrosine-phosphorylated binding
partner, the N-SH2 domain blocks the catalytic domain (20). Recently,
Lu et al. (21) presented a mechanism for the regulation of
SHP2 activity, suggesting that the N-SH2 domain interacts with the PTP
domain to inhibit phosphatase activity. SHP2 becomes activated by the
phosphorylation of tyrosine 542 or 580. Subsequently, these
phosphotyrosines interact with the N- and C-terminal SH2 domain,
respectively, relieving the PTP domain from the N-SH2 domain-mediated
inhibition (21). Binding of the SH2 domains to phosphopeptides deduced
from receptors or adapter molecules induces enzymatic activity
(22-24).
SOCS3 belongs to the family of suppressors of cytokine signaling (SOCS)
proteins. The members of this protein family (CIS and SOCS1 through
SOCS7) contain an N-terminal SH2 domain, preceded by the extended SH2
domain (ESS) and the kinase inhibitory region (KIR). Furthermore, a
homology domain called SOCS-box is located at the C terminus (25-30).
Primarily, SOCS1 and SOCS3 genes are rapidly induced by IL-6 and are
potent inhibitors of IL-6-mediated signaling. Thus, they are regarded
as classic feedback inhibitors (25-27). Several mechanisms by which
SOCS proteins inhibit cytokine signaling have been proposed: SOCS1, and
to some extent SOCS3, have been found to inhibit the kinase activity of
Jaks, probably by binding to the activation loop of the kinase (30,
31). Another model for SOCS function proposes the targeting of
signaling components to proteasome-dependent degradation
(32, 33). The latter is in line with the short half-lives of SOCS
proteins (34).
The contribution of SHP2 to tyrosine 759-dependent signal
attenuation has to be re-examined because we found that SOCS3 also acts
through and binds to the phosphotyrosine motif 759 of gp130 (35).
Actually, the affinity of SOCS3 to a phosphotyrosine peptide corresponding to the Tyr-759 motif of gp130 is much higher than to a
phospho-peptide comprising the activation loop of the Janus kinase
(36). Furthermore, the affinity of SOCS3 to bind gp130 is even slightly
higher than that of SHP2 (37). Although SOCS3·SHP2 complexes have
been observed, the interaction of SOCS3 and gp130 appears to be
SHP2-independent (35).
It was the goal of this study to dissect the contributions of SHP2 and
SOCS3 to Tyr-759-dependent signal attenuation. We asked whether SHP2 is involved in the Tyr-759-mediated repression of IL-6
signaling and whether SHP2 and SOCS3 act independently from each other.
Materials--
Restriction enzymes were purchased from Roche
Molecular Biochemicals (Mannheim, Germany) and Hybaid (Heidelberg,
Germany). Oligonucleotides were synthesized by MWG-Biotech (Ebersberg,
Germany). Vent polymerase and antibodies specific for activated STAT3
were obtained from New England BioLabs (Beverly, MA). Recombinant
erythropoietin (Epo) was a generous gift of Drs. J. Burg and
K. H. Sellinger of Roche Molecular Biochemicals (Mannheim,
Germany) and rIL-5 was purchased from Cell Concepts (Umkirch, Germany).
Antibodies to the extracellular domain of the IL-5R Construction of Expression Vectors--
Cloning was carried out
by standard procedures. pGL3 Transfection and Reporter Gene Analysis--
Human HepG2
hepatoma cells were grown and transiently transfected by the calcium
phosphate coprecipitation method as described previously (42).
Transfections were adjusted with control vectors to equal amounts of
DNA. Cell lysis and luciferase assays were carried out using the
luciferase kit (Promega, Madison, WI) as described by the manufacturer.
All transient expression experiments were done at least in triplicate.
Luciferase activity values were normalized to transfection efficiency
monitored by the cotransfected Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay--
The preparation of nuclear extracts, measurements of
protein concentrations, and EMSAs have been described previously (44). For STAT1- and STAT3-specific double-stranded 32P-labeled
probes, we used a mutated SIE oligonucleotide of the c-fos
promoter (m67 SIE, 5'-GATCCGGGAGGGATTTACGGGAAATGCTG-3') (45).
Protein·DNA complexes were separated on a 4.5% polyacrylamide gel
containing 7.5% glycerol in 0.25 × TBE at 20 V/cm for 4 h. Gels were fixed in 10% methanol, 10% acetic acid, and 80% water for
30 min, dried, and autoradiographed.
Immunoprecipitation and Immunoblot Analysis--
For
immunoprecipitations 2 × 107 cells were lysed in 500 µl of lysis buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 1 mM
EGTA; 1.5 mM MgCl2) or for peptide/protein coprecipitation in Brij-lysis buffer (1% Brij-96; 20 mM
Tris/HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA).
Buffers were supplemented with aprotinin, pepstatin, and leupeptin (10 µg/ml of each). Equal amounts of cellular protein were incubated with
the appropriate antibodies or with 2 µM biotinylated
peptides at 4 °C overnight and precipitated with 2.5 mg of Protein
A-Sepharose (Amersham Biosciences, Sweden) or NeutrAvidin-coupled
agarose (Pierce, Rockford, IL), respectively. Immune complexes
were separated by SDS-PAGE and transferred to a polyvinylidene
difluoride membrane. Antigens were detected by incubation with
the appropriate primary antibodies (PY99, 1:100; 4G10, 1:1000;
anti-SHP2, 1:1000; anti-Jak1, 1:1000; anti-FLAGM2-biotin, 1:500;
anti-Myc, 1:1000; IL-5R Expression of SOCS3 in Bacteria--
Human SOCS3 was expressed
as a thioredoxin fusion protein in BL21(DE3) Escherichia
coli (Stratagene, Heidelberg, Germany). Bacteria were grown in LB
medium containing 100 µg/ml ampicillin at 37 °C to an
A600 of 1 and then induced with 1 mM
isopropyl-1-thio- Biosensor Analysis--
Biotinylated peptides were loaded on a
streptAvidin-coated Biosensor chip (Biacore, Freiburg, Germany).
The amount of loaded peptide was 80 ± 4 fmol/mm2 chip
surface, which corresponds to 141 ± 5 response units. Before loading of the sensor chip with peptide, the surface was washed three
times for 30 s with 1 M NaCl/50 mM NaOH.
Peptides (100 ng/ml) were loaded onto the chip until 150 response units
were reached. Protein-peptide interactions were measured by injection
of serial dilutions of recombinant human SOCS3/thioredoxin fusion
protein over the chip surface at a flow rate of 20 µl/min for 1 min.
Before injection of SOCS protein, the sensor chip was flushed with
bovine serum albumin (0.1 mg/ml) at a flow rate of 20 µl/min for 1 min. For measurement of KD values, the flow rate was
enhanced to 100 µl/min to obtain higher resolution of kinetics. For
this type of experiment SOCS3 was injected for 3 min, dissociation time
was 5 min, and regeneration of the chip between the measurements in all
experiments performed was done at 20 µl/min with 1 M NaCl in 50 mM NaOH for 30 s. Binding curves were analyzed
by using BiaEvaluation software version 3.0.1 (Biacore). To correct for nonspecific binding events, an empty sensor surface without peptide was
analyzed in parallel during protein injection. Additionally, thioredoxin was injected at high concentrations (3.5 µM)
to rule out nonspecific interactions with the SOCS3 fusion protein.
Curves were plotted with nonspecific binding subtracted. Determination of dissociation constants was done by Scatchard analysis (46).
SHP2 Contributes to the Attenuation of Interleukin-6 Signal
Transduction--
The cytoplasmic tyrosine 759 of the signal
transducer gp130 mediates attenuation of IL-6 signaling (14-17). The
activated phosphotyrosine 759 functions as a recruitment site for the
tyrosine phosphatase SHP2 (10) and for SOCS3 (35, 36). To determine
whether SHP2 contributes to IL-6 signal attenuation, we analyzed
IL-6-induced STAT activation and promoter induction in murine
fibroblasts lacking exon 3 of SHP2. These cells express a mutant SHP2
protein lacking 65 amino acids within the N-terminal SH2 domain (48).
We checked whether this mutation affects recruitment to the tyrosine
motif 759 of gp130. Therefore, we analyzed binding of SHP2 to
corresponding un-phosphorylated (resembling non-activated receptors) or
phosphorylated (resembling activated receptors) gp130 receptor peptides
in wild-type fibroblasts as well as in SHP2-mut cells. Cellular
extracts of both types of cells were incubated with the indicated
biotin-conjugated peptides. Peptide·protein complexes were
precipitated with NeutrAvidin-coupled agarose and analyzed for presence
of SHP2 by Western blotting (Fig.
1A). Essentially no SHP2
binding was found in extracts of SHP2 mut cells (left
lanes), whereas SHP2 binding to the Y(p)759-peptide was apparent
in the wild-type cells (right lane).
To compare STAT activation in response to IL-6 these cells (SHP2 mut)
and corresponding wild-type fibroblasts (SHP2 wt) were stimulated with
IL-6·sIL-6R complexes. STAT3 DNA binding was analyzed by EMSA.
Stimulation of cells expressing mutated SHP2 led to enhanced and
sustained STAT3-DNA-binding activity when compared with wild-type cells
(Fig. 1B). We also tested whether the lack of wild-type SHP2
affects the activation of a STAT3-responsive promoter/reporter construct. Wild-type and mutant fibroblasts were transfected with a
chimeric EpoR/gp130 receptor construct, which allows stimulation independently of endogenous gp130. In line with the data above, activation of a promoter construct comprising STAT3-binding sites fused
to the luciferase reporter gene cDNA was enhanced in cells expressing mutated SHP2 (Fig. 1C). Mutated cells
reconstituted by stable expression of wild-type SHP2 did not show
enhanced reporter gene induction, similar to wild-type cells. These
data clearly indicate that a functional SHP2 attenuates IL-6-signal transduction.
Additionally, we analyzed whether expression of a catalytically
inactive form of SHP2 affects IL-6-mediated activation of downstream
signaling components. Mutation of the conserved asparagine (Asp-425) in
the catalytic domain of SHP2 is known to abolish enzymatic activity
(49). Myc-tagged SHP2 or SHP2D>A were expressed in COS-7 cells
together with an expression vector for Jak1 and chimeric receptors
containing the extracellular domain of the IL-5R
SHP2 and SHP2D>A and another catalytically inactive mutant SHP2C>S
were also analyzed for counteracting IL-6-induced STAT3 tyrosine
phosphorylation (Fig. 2B) and activation of the
liver-specific, STAT3-dependent
Targeted SHP2 Restores the Inhibitory Activity of gp130 Lacking the
Inhibitory Tyrosine 759--
The recruitment of SHP2 to
phosphotyrosine peptides is known to activate its phosphatase activity
(50). The underlying molecular mechanism was clarified by the
three-dimensional structure of SHP2, which showed that the SH2 domains
of inactive SHP2 cover the catalytic domain, whereas binding to
phosphopeptides leads to a conformational change and activation of the
enzyme (20). Deletion of the SH2 domains results in the formation of an
active phosphatase, which cannot be recruited to phosphotyrosine motifs of activated receptors (23, 51). Signal transduction from gp130
containing a mutated tyrosine 759 is enhanced, because the receptor
lacks the inhibitory SHP2·SOCS3 recruitment site. We have shown that
the inhibitory tyrosine 759 of gp130 does not have to be located on the
same receptor chain as the STAT-binding sites to exert its inhibitory
action (17).
We asked whether forced recruitment of SHP2 to Y759F-mutated gp130
could restore the inhibitory activity in IL-6-induced signal transduction. Therefore, we fused the C-terminal part of SHP2 containing the tyrosine phosphatase domain (PTP) to gp130 lacking tyrosine 759. To maintain STAT activation, the C-terminal part of SHP2
was fused only to a single receptor chain of the gp130 dimer. Again, we
used combinations of IL-5R/gp130 chimeric receptors, which allowed the
heterodimerization of different cytoplasmic domains of gp130 through
stimulation with IL-5. Flow cytometric analyses were performed to
monitor whether mutations in the chimeric IL-5R
Interestingly, signal transduction through a receptor complex
containing a receptor-fused SHP2-PTP domain was reduced
((IL-5R
The potential of the SHP2-PTP domain to restore the inhibitory activity
of gp130 mutants lacking Tyr-759 should be confirmed with an
independent experimental approach. Therefore, we checked whether a
membrane-anchored SHP2-PTP domain also restores the inhibitory
activity. For this purpose, the short membrane-anchoring signal of the
Src-kinase hck was fused to the N terminus of the C-terminal SHP2
fragment. PTPs without (PTP) or with a membrane anchor ( The Inhibitory Activity of SOCS3 Depends on the Presence of
Tyrosine 759 of gp130 but Not on Functional SHP2--
As shown in Fig.
1 (B and C), lack of functional SHP2 leads to
enhanced signal transduction (as measured by STAT activation and
reporter gene induction), similar to the effect of a mutated Tyr-759 in
gp130. The inhibitory activity of SOCS3 also depends on Tyr-759 in
gp130. Thus, we asked whether SOCS3 function depends on the presence of
wild-type SHP2. We compared reporter gene activation through
EpoR/gp130(YYYYYY) and EpoR/gp130(YFYYYY) in the fibroblast
cell lines expressing wild-type or mutated SHP2. Stimulation of
fibroblasts expressing the wild-type SHP2 led to a 5-fold induction of
the reporter gene (1 in Fig.
4A). In these cells the Y759F
exchange in the gp130 part of the chimeric receptors led to an enhanced
induction of the reporter gene as already shown above for HepG2 cells
(Fig. 4A, compare 1 and 3). Again, the
inhibitory activity of SOCS3 appeared to depend on the presence of
Tyr-759 in gp130 (compare 1 and 2 with
3 and 4). As shown in Fig. 4B, the
presence of a functional SHP2 did not appear to be crucial for SOCS3
inhibitory activity, because SOCS3 acts inhibitory on non-mutated
chimeric receptors in both cell lines. The presence of Tyr-759 in gp130 remains to be essential for SOCS3 action in both cell lines (compare 3 and 4 in Fig. 4, A and
B). In summary, the inhibitory activity of SOCS3 depends on
the presence of tyrosine 759 of gp130 but not on functional SHP2.
The Extended SH2 Domain of SOCS3 Is Essential for the Interaction
with gp130 but Dispensable for the Interaction with SHP2--
An
interaction between SHP2 and SOCS3 was already known from our previous
studies (35). The nature and function of these SHP2·SOCS3 complexes,
however, remained unclear. A contribution to signal attenuation is
quite possible.
To analyze the binding of SOCS3 to SHP2 in greater detail, we prepared
SOCS3 deletion mutants, lacking the C-terminal SOCS-box (S3 SOCS3 Interacts with the Y(p)542 Module of SHP2--
The
SHP2·SOCS3-binding site Y(p)759 of gp130 (VQpYSTVH) and the Y(p)542
motif of SHP2 (HEpYTNIK) display some structural and functional
similarities. SHP2 binds to the tyrosine motif of gp130 (10),
additionally, the N-terminal SH2 domain of SHP2 interacts intramolecularly with the phosphotyrosine 542 motif of SHP2 to overcome
autoinhibition (21). SOCS3 also binds to the Y(p)759 of gp130 (35, 36).
Thus, we speculated that SOCS3 may bind SHP2 through the Y(p)542 motif
of SHP2. Indeed, peptide precipitations in Fig.
6A show that SOCS3 binds both
the Y(p)759-peptide, derived from gp130 and the Y(p)542-peptide from
SHP2, but not the non-phosphorylated Tyr-542-peptide or unrelated
peptides from gp130 (Tyr-683 and Y(p)683) or SHP2 (Y580 and Y(p)580).
To further compare binding of SOCS3 to the gp130 and SHP2 peptides,
biosensor analyses were performed with recombinant human SOCS3
thioredoxin fusion proteins. Fig. 6B shows that SOCS3
specifically binds the Y(p)759 motif of gp130 and the Y(p)542 peptide
of SHP2. Hardly any binding could be detected using the Y(p)580 of SHP2
or the unphosphorylated Tyr-542 peptide. To compare the dissociation
constants for the binding of SOCS3 to Y(p)542 of SHP2 and to Y(p)759 of
gp130, binding was analyzed in the presence of decreasing amounts of
SOCS3 (Fig. 6, C and E, respectively).
KD values derived from Scatchard plots (Fig. 6,
D and F) were 210 nM for the
SOCS3/Y(p)759 interaction and 3.5 µM for binding the
Y(p)542 peptide of SHP2. Thus, the affinity of SOCS3 to the receptor
peptide appears to be 17 times higher than to the SHP2-peptide.
Nevertheless, a remarkable affinity was found for the binding of SOCS3
to the phosphotyrosine 542 motif of SHP2.
SHP2 Targeting to Receptors Lacking the SHP2·SOCS3 Recruitment
Site Tyr-759 Does Not Restore Sensitivity to SOCS3--
Mutation of
Y759F in gp130 affects gp130-dependent promoter activation
as well as SHP2 recruitment (10, 14-17). Furthermore, gp130 mutated at
Tyr-759 is not sensitive to inhibition by SOCS3 and does not bind SOCS3
(35, 36). We asked whether targeted SHP2 also restores sensitivity to
SOCS3 in receptor complexes lacking the SHP2·SOCS3 recruitment site.
The effect of SOCS3 expression on
Similar results were obtained with membrane-anchored SHP2 (Fig.
7B). Signal transduction through chimeric EpoR/gp130 lacking Tyr-759 is not affected by coexpression of SOCS3 but by coexpression of
membrane-anchored SHP2 (
From these data we suggest that targeted SHP2 can not restore
inhibitory activity of SOCS3 on gp130-(Y759F)-mutated receptors suggesting that the interaction of SHP2 with SOCS3 is not involved in
signal attenuation. Thus, the physiological relevance of this interaction has to be clarified by further experiments.
Modulation of ERK Activity through SOCS3 Mutants--
Tyrosine 759 of gp130 is also essential for the activation of the MAPK cascade.
Mutation of tyrosine 759 to phenylalanine was shown to abolish
IL-6-mediated activation of Erk1/Erk2 (55). It has been suggested that
recruitment of SHP2 to this phosphotyrosine 759 motif mediates
activation of Erk1/Erk2. Thus, one potential function of the
SHP2·SOCS3 complex might be to modulate IL-6-induced MAPK activity.
Therefore, SOCS3 mutants with different potential to bind gp130 and
SHP2 were analyzed to influence IL-6-induced MAPK activation
independent of endogenous SOCS3. Epo-induced MAPK activity was
monitored by using an Elk1 transactivation approach (PathDetect Elk1
trans reporting system, Stratagene) (Fig.
8). Because activation of
EpoR/yp130(YYFFFF) chimeric receptors did not lead to
STAT3-dependent gene induction, these receptors enabled us
to analyze MAPK activation independently of the induction of endogenous
SOCS3. Indeed, MAPK activation was more pronounced through
EpoR/yp130(YYFFFF) than through Epo/gp130(YYYYYY) (compare
panels 1 and 3 in Fig. 8) suggesting that SOCS
induction through the wild-type receptor negatively affects MAPK
activation. The increased MAPK activity through receptors lacking
STAT-binding sites (EpoR/yp130(YYFFFF)) is likely due to the
lack of SOCS induction through this receptor. This is further supported by the observation that exogenous SOCS3 reduces MAPK activity (compare
panels 3 and 4). Interestingly, SOCS3(R71K)
inhibits MAPK activation (compare panels 3 and
7), although this SOCS3 mutant does not bind to the receptor
but SHP2 (Fig. 5). SOCS3 (R71E), which could neither be precipitated
with the receptor peptide Y(p)759 nor with SHP2, did not act inhibitory
on MAPK activation (compare panels 3 and 8). The
SOCS3 proteins with mutations within the KIR (SOCS3F25A) and the ESS
(SOCS3L41R) also show impaired inhibitory activity (compare panel
3 with panels 5 and 6). Whether these
observations indicate an alternative mechanism for inhibition of MAPK
activity by SOCS3 independent of direct receptor binding remains to be
clarified by further studies.
After stimulation with IL-6, the protein-tyrosine phosphatase SHP2
is recruited to the phosphotyrosine motif 759 of gp130 within the
activated receptor complex (10). The observation that the mutation of
tyrosine 759 to phenylalanine enhances IL-6 signal transduction led to
the assumption that SHP2 mediates an inhibitory activity (14-16, 18).
The contribution of SHP2 to tyrosine 759-dependent signal
attenuation had to be re-examined, because we found out that SOCS3 also
acts by binding to the phosphotyrosine 759 motif of gp130 (35). This
observation was confirmed by affinity measurements showing a higher
affinity of SOCS3 to the phosphorylated Tyr-759 receptor peptide than
to peptides corresponding to the activation loop of the Janus kinases
Jak1, 2, or 3 (36).
Here we demonstrate that SHP2 indeed affects IL-6 signaling, because
stimulation of cells lacking full-length SHP2 results in an increased
gp130-mediated signal transduction as measured by STAT activation and
STAT3-dependent gene induction (Fig. 1). In addition,
expression of a catalytically inactive form of SHP2 leads to an
increase in receptor, Jak, and STAT3 phosphorylation as well as gene
induction (Fig. 2). Furthermore, the inhibition of signal transduction
in cells expressing gp130 mutants lacking the SOCS3/SHP2 recruitment
site in gp130 can be restored by membrane targeting of the C-terminal
part of SHP2 containing the PTP domain and also by fusion of this
polypeptide to the membrane-proximal part of gp130 (Fig. 3). These data
indicate an inhibitory function of SHP2 on IL-6 signaling independent
from receptor-bound SOCS3. On the other hand, SOCS3 action does not
depend on functional SHP2, because cells expressing only mutated SHP2
remain sensitive to SOCS3, but become insensitive, when the SOCS3/SHP2
recruitment site in gp130 is mutated (Fig. 4).
We observed that SOCS3 binds in a different manner to receptor peptides
and SHP2 (Fig. 5). Binding to tyrosine 759 of gp130 was highly
sensitive to mutations within the ESS and the SH2 domain of SOCS3. In
clear contrast, only the substitution of the crucial arginine within
the SH2 domain of SOCS3 by glutamate impaired binding of SOCS3 to SHP2.
Furthermore, SOCS3 binds the phosphotyrosine peptide derived from
the Tyr-759 motif of gp130 with a 17-fold higher affinity than
the peptide derived from the Tyr-542 motif of SHP2 (Fig. 6). These
affinities may also reflect the more restrictive requirements for
binding to gp130. Whether this interaction is really of major relevance
for SOCS3 function remains to be further elucidated.
From all these data, we suggest that there are two, largely distinct
modes of negative regulation of gp130 activity, and probably of other
cytokine receptors, despite the fact that both SOCS3 and SHP2 are
recruited to the same site within gp130. Binding to the same receptor
site implies competitive binding of both proteins to gp130, which would
require SHP2 and SOCS3 being present at the receptor at the same time.
This seems to be possible, because SOCS3 protein becomes detectable
already after 20 min, at a time where SHP2 is still phosphorylated
(data not shown). Tyrosine phosphorylation was recently clearly
linked to enzymatic activity of SHP2 (21). Because SOCS3 protein
remains longer detectable than SHP2 phosphorylation, SHP2 may be
important for early signal modulation, whereas SOCS3 has to be induced
and probably attenuates signaling subsequently. Furthermore, it is
intriguing to speculate in terms of cross-talk, assuming that SOCS3 may
also be induced by other cytokines prior to IL-6-stimulation. As a
consequence SOCS3 would be immediately available to block IL-6
signaling and also to compete with SHP2 for receptor binding.
Similar to our results for the signal transduction of IL-6, it has been
shown that SOCS3 and SHP2 are recruited to the same receptor motifs
within the leptin receptor. Furthermore, the inhibitory function of
SOCS3 depends on this SOCS2/SHP2 binding site (56, 57). In contrast to
the data for IL-6 signaling presented here, it has been suggested that
SOCS3 but not SHP2 is involved in attenuation of leptin signaling.
Instead, SHP2 was attributed to mediate MAPK activation (56, 58).
Tyrosine 759 of gp130 is also essential for the activation of the
MAPK cascade (55). The current view of this activation, adopted from
epidermal growth factor and platelet-derived growth factor signal
transduction, is that SHP2 links the Grb2·SOS complex and/or Gab1/2
to gp130 (55, 59, 60). Additionally, it has been found that
IL-6-induced association of the adapter protein Gab1 with SHP2 also
leads to an activation of the MAPK cascade (61). Recently, Cacalano
et al. (62) suggested that the MAPK cascade can also be
affected through SOCS3: in response to IL-2, Epo, and platelet-derived
growth factor, SOCS3 becomes tyrosine-phosphorylated and subsequently
binds and inactivates Ras/GAP. The latter leads to a sustained Ras
activation and consequently to activation of the MAPK cascade. We
presented the first evidence that SOCS3 might also modulate MAPK
activation after stimulation with IL-6. It is further intriguing to
speculate that SOCS3 may specifically inhibit the Jak/STAT pathway
while maintaining MAPK activation.
Meanwhile, several other cytokine and growth factor receptors have been
described to bind SOCS3 as well a SHP2 (47, 56). For each of these
receptors the individual role of SHP2 and SOCS3 needs to be analyzed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (IL-6R
, gp80, or CD126) and the signal
transducing subunit gp130 (CD130) (6, 7). Ligand binding to the
-subunit leads to the dimerization of gp130 and the activation of
the constitutively associated Janus kinases Jak1, Jak2, and Tyk2 (8,
9). In turn, gp130 becomes tyrosine-phosphorylated on its cytoplasmic tail and recruits transcription factors of the family of signal transducers and activators of transcription (STAT), STAT1 and STAT3, to
specific phosphotyrosine motifs (10, 11). Subsequently, STAT factors
become tyrosine-phosphorylated, dissociate from the receptor complexes,
and translocate to the nucleus, where STAT homo- and/or heterodimers
bind to specific DNA elements in the promoters of IL-6 target genes
(12, 13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain were
generously provided by Jan Tavernier (Gent, Belgium). Peptides were
prepared according to standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) synthesis protocols. The
peptides used had the following amino acid sequences: gp130:
(Y(p)683, biotin-
A-NSKDQMpYSDGNFTD; Tyr-759,
biotin-
A-TSSTVQYSTVVHSG; and Y(p)759, biotin-
A-TSSTVQpYSTVVHSG),
SHP2: (Tyr-542, biotin-
A-KRKGHEYTNIKYSL; Y(p)542,
biotin-
A-KRKGHEpYTNIKYSL; Tyr-580, biotin-
A-EDSARVYENVGLMQ, and Y(p)580, biotin-
A-EDSARVpYEN- VGLMQ) (pY indicates
phosphotyrosine). The phosphotyrosine-specific antibody PY99 and
antibodies to SHP2, to Jak1, and to the extracellular region of the
IL-5R
-chain were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). For immunoprecipitation of Jak1 a polyclonal antibody raised
against Jak1 was kindly provided by A. Ziemiecki (Bern). The
phosphotyrosine antibody (4G10) and the gp130 antibody were from
Upstate Biotechnology Inc. (Lake Placid, NY), and FLAG tag
antibodies were from Sigma Chemical Co. (St. Louis, MO) and the Myc tag
antibodies were from Roche Molecular Biochemicals (Mannheim, Germany).
Recombinant IL-6 and sIL-6R were prepared as described previously (38).
The specific activity of IL-6 was 2 × 106 B
cell-stimulatory factor (2 units/mg of protein).
2M-215Luc contains the
promoter region
215 to +8 of the rat
2-macroglobulin fused to the luciferase encoding sequence and was described previously (15). The SIE-tk-Luc construct, containing two copies of the STAT
consensus binding sequence from the c-fos promoter upstream of a thymidine kinase minimal promoter (39), was kindly provided by
Hugues Gascan (Angers, France). Expression vectors for the IL-5R/gp130
receptor chimeras pRcCMV-IL-5R
/gp130(YYYYYY),
pRcCMV-IL-5R
/gp130(YYYYYY), pRcCMV-IL-5R
/gp130(YFYYYY),
pRcCMV-IL-5R
/gp130(YFYYYY), pRcCMV-IL-5R
/gp130(YYFFFF), and
pRcCMV-IL-5R
/gp130(YFFFFF) were used in reporter gene
assays as described previously (17). pRcCMV-IL-5R
/gp130(YF) encodes a C-terminal deletion
mutant of IL-5R
/gp130(YFYYYY) lacking amino acids
766-918 of gp130. For construction of the
pRcCMV-IL-5R
/gp130(YF)/PTP chimera, the cDNA of an SHP2
protein lacking the N-terminal 208 amino acids was amplified by PCR.
This fragment was fused to the 3'-end of the cDNA of the
IL-5R
/gp130(YF) vector by simultaneous elimination of the
stop codon. For steric flexibility a linker sequence of glycine and
serine residues (GS)5G was introduced between the residual
part of gp130 and the SHP2 fragment giving pRcCMV-IL-5R
/gp130(YF)/PTP. The construction of
pCBC1-SHP2WT and pCBC1-SHP2C>S was carried out as previously described
(15). PCBC1-SHP2D>A was generated to encode a gene product that
contains an Arg to Ala exchange at position 425 in SHP2. Vector
pCBC1-
-PTP encodes a mutant of human SHP2 lacking both SH2 domains
(amino acids 1-208). For membrane targeting the myristoylation signal MGCMKSKFLQ of the murine hematopoietic cell kinase (hck)
proto-oncogene p59 was fused to the N terminus. The expression vector
pCBC1-PTP is identical to pCBC1-
-PTP but lacks the myristoylation
signal. These constructs were cotransfected for reporter gene assays
with previously described EpoR/gp130 receptor chimeras
(pRcCMV-EG(YYYYYY) and pRcCMV-EG(YFYYYY)) (15). Jak1
expression vector (pSVL-Jak1) was described previously (40). The
expression vector for murine SOCS3 was pEF-FLAG-I/mSOCS3 (26), kindly
provided by D. Hilton, Melbourne, Australia. The SOCS3 mutants
S3
box, S3
N-term, and S3
ESS were generated by exchange of the
full-length SOCS3-cDNA by of DNA fragments coding for amino acids
1-183, 23-225, or 44-225 of SOCS3, respectively. The SOCS3 deletion
mutants S3
N20, S3
N36, S3
C40, S3
C84, and point mutations
S3L22D, S3F25A, S3L41R, S3G45A, S3R71K, and S3R71E were described
previously (41) (S3 stands for SOCS3).
-galactosidase expression vector
(pCR3lacZ, Amersham Biosciences, Uppsala, Sweden) (1.5 µg). COS-7
cells were grown in DMEM with 10% FCS, 100 mg/liter streptomycin, and
60 mg/liter penicillin. Approximately 1.5 × 107 COS-7
cells were transiently transfected with 6-25 µg of DNA using the
DEAE-dextran method. Briefly, cells were incubated in medium containing
the DNA, 80 µM chloroquine, and 0.4 mg/ml DEAE-dextran for 80 min avoiding gas exchange. Afterward, cells were incubated for 1 min in phosphate-buffered saline containing 10% Me2SO.
After 24 h cells were split 1:2, and, after additional 24 h
in culture medium, cells were stimulated. 3T3 embryonal fibroblasts
were from SHP2 wild-type mice (SHP2 wt) or SHP2 exon 3-deficient mice (SHP2-mut) or reconstituted SHP2-mut cells stably expressing wild-type SHP2 were grown in DMEM with 15% FCS, 100 mg/liter streptomycin, and
60 mg/liter penicillin (43). These cells were transiently transfected
with FuGENE 6 transfection reagent (Roche Molecular Biochemicals,
Mannheim, Germany) as described in the manufacturer's instructions.
MAPK activity was monitored by GAL4/ELK-reporter analyses. Experiments
were performed as described by Stratagene's instructions for the
PathDetect Elk1 trans reporting system (Stratagene Europe,
Amsterdam). Briefly, luciferase reporter pFR-Luc containing a
Gal4-promoter element was cotransfected with an expression vector for a
fusion protein composed of the DNA-binding domain of Gal4 and the
transactivation domain of the Elk1 transcription factor (pFA2-Elk1).
Transfection efficiency was monitored by using a cotransfected
-galactosidase expression vector as described above. Activated MAPKs
lead to phosphorylation of the Elk transactivation domain and thus to
expression of luciferase.
, 1:1000; and IL-5R
, 1:1000) and
horseradish peroxidase-coupled secondary antibodies (1:2000) (Dako,
Hamburg, Germany) or horseradish peroxidase-coupled streptavidin
(1:5000) (Pierce, Rockford, IL). The membranes were developed with an
enhanced chemiluminescence kit (Amersham Biosciences, Freiburg,
Germany). Blots were stripped and reprobed to verify application of
equal amounts of protein.
-galactopyranoside. Cells were harvested after
3 h of expression, resuspended in 50 mM Tris/HCl, pH
8.0, 10% glycerol, and lysed by sonification. SOCS3 was purified on a
HiTrap chelating 5-ml column (Amersham Biosciences, Freiburg, Germany)
with nickel-iminodiacetic acid (Ni-IDA) as matrix. Native eluted
SOCS3 was dialyzed into 50 mM Tris, 10 mM
dithiothreitol, pH 8.5 and purified to homogeneity by anion-exchange
chromatography on a Mono Q column (Amersham Biosciences, Freiburg,
Germany). For biosensor measurements the protein was dialyzed against
50 mM Tris/HCl, pH 8.0, 10 mM dithiothreitol, 0.05% Chaps. Purity of the recombinant protein was monitored by SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SHP2 is involved in negative regulation of
IL-6 signal transduction. A, cellular extracts of
murine embryonal 3T3 fibroblast from SHP2-Exon 3 (SHP2
mut)-deficient mice or corresponding wild-type cells (SHP2
wt) were incubated with biotinylated peptides Tyr-759 or Y(p)759
corresponding to the SHP2-binding site in gp130. After precipitation
with NeutrAvidin-coupled agarose the proteins were subjected to Western
blot analyses (IB) using antibodies to SHP2. B,
STAT DNA binding in SHP2 wt and SHP2 mut cells after stimulation with
20 ng/ml IL-6 and 1 µg/ml sIL-6R for times indicated was analyzed by
EMSA with STAT3-specific DNA probes. Bands resulting from STAT3
homodimers bound to the DNA probe are indicated by an
arrowhead. C, wild-type cells, SHP2 mut cells and
corresponding SHP2-reconstituted cells were transiently transfected
with expression vectors coding for chimeric EpoR/gp130 receptors
(containing the extracellular part of the EpoR and the transmembrane
and cytoplasmic part of gp130) and the SIE-tk-luciferase reporter gene
construct (3 µg). The cells were stimulated for 16 h with 7 units/ml Epo as indicated, and cellular extracts were prepared for the
determination of luciferase activity. Luciferase expression was
normalized to the luciferase activity in cell extracts from stimulated
wild-type cells. Data shown are the averages of triplicate independent
determinations of firefly luciferase activity normalized to
-galactosidase activity ± S.E. and are representative of
multiple similar experiments.
or IL-5R
and the
transmembrane and cytoplasmic domains of gp130. These receptor chimeras
allowed us to study the IL-6 signal transduction independently from
endogenous gp130 (17). After stimulating the cells with IL-5, lysates
were prepared and immunoprecipitations with antibodies to IL-5R
,
Jak1, or the Myc-tag of SHP2 and SHP2D>A were carried out. The
phosphorylation status of the precipitated proteins was detected in
Western blots with phosphotyrosine-specific antibodies (Fig.
2A). As shown in the upper panel of Fig. 2A, stimulation of COS cells
expressing wild-type SHP2 led to increased Jak1, IL-5R
/gp130, and
SHP2 tyrosine phosphorylation. The expression of SHP2D>A further
increased the induced as well as the basal phosphorylation of these
signaling molecules. Finally, we observed, that a high percentage of
phosphorylated Jak1 coprecipitates with SHP2D>A, which may indicate
Jak1 as a direct substrate for SHP2.
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Fig. 2.
SHP2 is involved in negative regulation of
IL-6 signal transduction. A, COS-7 cells were
cotransfected with expression vectors for IL-5R /gp130 and
IL-5R
/gp130 (6 µg each), Jak1 (0.8 µg), and SHP2 or SHP2D>A (12 µg). 40 h after transfection the cells were starved with DMEM
without FCS for 8 h. Cells were stimulated for 20 min with 10 ng/ml IL-5, and cellular extracts were prepared. Lysates were incubated
with indicated antibodies and protein·antibody complexes were
separated by SDS-PAGE and analyzed by Western blotting with the
indicated antibodies (IB). M, protein marker.
B, COS-7 cells were transfected with expression vectors for
SHP2, SHP2D>A, or SHP2C>S (10 µg), IL-5R
/gp130 and
IL-5R
/gp130 (6 µg each), Jak1 (0.8 µg), and STAT3 (3 µg).
40 h after transfection, the cells were stimulated with 80 ng/ml
IL-5 of left untreated. 30 min post stimulation cells were harvested,
and lysates were prepared. The same amounts of protein (130 µg) were
separated by SDS-PAGE and analyzed by Western blotting with the
antibodies for activated STAT3 (upper panel) and after
stripping of the blot with antibodies for STAT3 as a loading control.
C, HepG2 cells were cotransfected with expression vectors
encoding EpoR/gp130 chimeric receptors (2 µg), the
2M-215 luciferase reporter-gene construct (3 µg), and
SHP2, SHP2C>S, or SHP2D>A (4 µg). The cells were stimulated for
16 h with 4 units of Epo/ml as indicated, and cellular extracts
were prepared for the determination of luciferase activity. Luciferase
expression was normalized to the luciferase activity in cell extracts
from stimulated cells transfected with wild-type SHP2. Data are given
as means ± S.E. of at least three independent experiments.
2-macroglobulin promoter (Fig. 2C). SHP2,
SHP2D>A, and SHP2C>S were expressed in COS-7 cells similarly as
described for Fig. 2A. The cells were stimulated with IL-5,
cell lysates were prepared, and STAT3 activation was analyzed by
Western blotting with antibodies specific for tyrosine
705-phosphorylated STAT3 (Fig. 2B). As shown in the
upper panel of Fig. 2B expression of inactive
SHP2 mutants led to increased basal and induced STAT3 activation,
similarly to the observations made for Jak1 and the receptor in Fig.
2A. Additionally, the same amounts of expression vector for
these three SHP2 constructs, together with an
2M-promoter/luciferase reporter and cDNA for
chimeric EpoR/gp130 receptors (composed of the extracellular domain of
the EpoR and the transmembrane and cytoplasmic domains of gp130) were
expressed in human hepatoma cells (HepG2) (Fig. 2C).
Stimulation of HepG2 cells expressing wild-type SHP2 led to an increase
in promoter activity. Expression of the two enzymatically inactive SHP2
forms, where the conserved Asp-425 or Cys-459 residues were mutated to
Ala or Ser, respectively, further increased reporter gene activity to
at least 2-fold. Thus, mutations within the catalytic domain of SHP2
counteract the inhibitory activity of SHP2. The results from both
experimental approaches indicate an inhibitory function of SHP2 for
interleukin-6 signal transduction.
/gp130 receptors
affected cell surface expression. Whereas the mutation of single
tyrosine residues in gp130 did not alter receptor expression
significantly (Fig. 3A, flow
cytometric profiles on the left panel), deletion of the 153 C-terminal amino acids of gp130 led to an enhanced surface expression
(right panel), probably through the loss of the
internalization signal in gp130 as previously observed (52-54). As
shown in Fig. 3B, mutation of Tyr-759
((IL-5R
/gp130(YFYYYY) · IL-5R
/gp130(YFYYYY)) led to an increased promoter
activation (compare 1 and 2). Tyrosine 759 also
inhibits reporter expression when located in trans to the
STAT recruitment sites on the second receptor chain
((IL-5R
/gp130(YYFFFF) · IL-5R
/gp130(YFYYYY)) (compare 3 and
4). Analysis of reporter gene induction through
(IL-5R
/gp130(YF) · IL-5R
/gp130(YFYYYY)) (panel 5) shows that
the C-terminal part of (IL-5R
/gp130(YFFFFF) (panel
4) does not contribute to signal transduction.
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Fig. 3.
Targeted SHP2 restores the inhibitory
activity of gp130 mutants lacking the inhibitory tyrosine 759. A, surface expression of the IL-5R /gp130 chimeras and
IL-5R
(YF-PTP) fusion proteins was monitored by FACS analyses in
transiently transfected COS-7 cells, using an antibody raised against
the extracellular part of the IL-5R
(open histograms).
Filled histograms show FACS controls with secondary antibody
only. B, human hepatoma cells (HepG2) were transfected with
equal amounts (2 µg) of expression vectors coding for chimeric
receptors as indicated in the figure and an
2M-promoter-luciferase reporter construct
(pGL3
2M-215Luc). The cells were stimulated for 16 h
with 10 ng/ml human recombinant IL-5 where indicated. Cellular extracts
were prepared for determination of luciferase activity, which was
normalized to the activity of coexpressed
-galactosidase. Luciferase
activity is presented in relation to the activity in cellular extracts
from stimulated HepG2 cells expressing the IL-5R
- and IL-5R
-gp130
wild-type chimeric receptors. Data are given as means ± S.D. of
at least three independent experiments. C, HepG2 cells were
transiently transfected with 20 µg of expression vectors for PTP,
-PTP, or the corresponding control plasmid. 48 h post
transfection cells were harvested, and proteins were separated by
SDS-PAGE. Western blots were developed with SHP2 antibodies.
D, HepG2 cells were transfected with 2 µg of expression
vectors for the EpoR/gp130 chimeric proteins EG(YYYYYY) or
EG(YFYYYY) together with 4 µg of expression vectors for
PTP,
-PTP, or the corresponding control vector and the
2M-promoter-luciferase reporter construct. Cells were
stimulated for 16 h with 7 units/ml Epo, lysed, and assayed for
luciferase activity as described above.
/gp130(YF)/PTP · IL-5R
/gp130(YFYYYY)) (compare 5 and
6) despite the higher expression of this fusion protein. In
summary, receptor-targeted SHP2-phosphatase activity, at least
partially, restores the inhibitory activity of a mutated tyrosine 759 motif in gp130.
-PTP) were
expressed in HepG2 cells. Expression of both proteins appeared to be
similar as detected by Western blot analyses (Fig. 3C). IL-6
signal transduction in the presence of these proteins was analyzed by
activating coexpressed chimeric EpoR/gp130 receptors with Epo. As
expected, induction of an
2M-promoter/reporter through receptors lacking Tyr-759 was enhanced when compared with receptors containing the cytoplasmic part of the wild-type gp130 (compare 1 with 2, Fig. 3D). Whereas expression
of non-anchored PTP hardly affects reporter gene activity (compare
2 and 3) anchored
-PTP inhibits promoter
activation mediated through receptors lacking tyrosine 759 (compare
2 and 4). These experiments further demonstrate
that targeted SHP2 is able to restore the inhibitory activity of
Tyr-759-mutated gp130 lacking the SHP2·SOCS3 recruitment site.
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Fig. 4.
Functional SHP2 is not required for
inhibition by SOCS3. SHP2 wt (A) and SHP2 mut cells
(B) were transiently transfected with 2 µg of expression
vectors coding for EG(YYYYYY) or EG(YFYYYY), 0.25 µg of
SOCS3-cDNA where indicated and 2 µg of a SIE-tk-luciferase
reporter gene. The cells were stimulated for 16 h with 7 units/ml
Epo as indicated, and cellular extracts were prepared for the
determination of luciferase activity. Luciferase expression was
normalized to the luciferase activity in cell extracts from stimulated
cells expressing EG(YYYYYY). Data are given as means ± S.E. of at
least three independent experiments.
box), the
23 N-terminal amino acids (S3
N-term), or, in addition to the latter,
a region identified as an extension of the SH2 domain (28, 30)
(S3
ESS) (Fig. 5A). These
proteins were transiently expressed in COS-7 cells (Fig. 5B)
and tested for SHP2 binding using coimmunoprecipitation assays (Fig.
5D). As a control, binding of these proteins to the
phosphotyrosine peptide Y(p)759 of gp130 was monitored by peptide
precipitation assays (Fig. 5C). Fig. 5C shows
that neither the SOCS-box nor the N-terminal 23 amino acids of SOCS3 is
essential for binding to Y(p)759. However, the extended SH2 domain
(ESS) of SOCS3 is essential for binding to the receptor peptide
Y(p)759. Binding to SHP2 appeared to be independent of the ESS region,
because none of these mutations impaired binding to SHP2 (Fig.
5D, upper panel). Another set of SOCS3 mutants
with C- and N-terminal deletions or single point mutations was used to
further map the region responsible for SHP2-binding (Fig.
5E). Deletion of the N-terminal 36 amino acids (S3
N36) as
well as point mutations within the ESS (S3L41R) and within the SH2
domain (S3R71K; S3R71E) of SOCS3 impaired binding to the receptor
peptide (Fig. 5F, upper panel). Only the latter
mutant within the SOCS3-SH2 domain impaired SOCS3·SHP2 complex
formation (Fig. 5F, lower panel). These data
demonstrate that binding of SOCS3 to gp130 or to SHP2 depends on the
integrity of the SOCS3-SH2 domain. Different modes of interaction have
to be assumed, because binding of SOCS3 to the receptor peptide is
affected by the exchange of Arg-71 to Lys but binding to SHP2 is only
impaired by exchange to Glu. Additionally, these experiments
demonstrate that receptor binding requires the ESS of SOCS3, because
deletion of the ESS as well as a single point mutation (L41R) within
the ESS of SOCS3 impairs its binding to the gp130 receptor peptide but
not to SHP2.
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Fig. 5.
SOCS3 interacts with SHP2 through its SH2
domain. A, representation of the SOCS3 mutants used.
B-F, COS-7 cells were transfected with expression vectors
(10 µg) for SOCS3, SOCS3 deletion- or point mutants, as indicated.
48 h after transfection and cultivation in FCS-containing DMEM
cellular extracts were prepared for immunoprecipitation (IP)
or peptide precipitation (PP). B, to verify the
expression of the SOCS3 mutants, immunoprecipitation with antibodies
against the FLAG-tagged SOCS3 proteins was performed. Bound proteins
were separated by SDS-PAGE and analyzed by Western-blot for SOCS3.
C, cells transfected with the indicated SOCS3 mutants were
lysed and incubated with biotinylated peptides Tyr-759 or Y(p)759.
After precipitation with NeutrAvidin-coupled agarose the proteins were
subjected to Western-blot analyses (IB) using antibodies to
SHP2 (lower panel) or to the FLAG tag of the SOCS3 proteins
(upper panel). D, the SOCS3 mutants used in
B and C were also tested for binding to SHP2.
Binding of SHP2 to these SOCS3 mutants was tested by
coimmunoprecipitation with an antibody raised against SHP2. Bound
proteins were separated by SDS-PAGE and analyzed for SHP2 (lower
panel) and coprecipitated SOCS3 (upper panel) by
Western blotting (IB). E, the expression of SOCS3
mutants was analyzed by Western blotting of cellular extracts described
for D. F, peptide precipitation of the indicated
SOCS3 mutants (upper panel) or endogenous SHP2 (middle
panel) with the biotinylated Y(p)759 peptide was performed as
described for C. The association of SOCS3 mutants with SHP2
were determined as described in D (lower
panel).
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Fig. 6.
SOCS3 binds to the Y(p)542-peptide of SHP2
with lower affinity than to the Y(p)759-peptide of gp130.
A, cellular extracts of COS-7 cells expressing SOCS3 were
prepared and incubated with the biotinylated peptides as indicated
(PP). After precipitation with NeutrAvidin-coupled agarose,
the proteins were subjected to Western blot analysis (IB)
using antibodies to the FLAG tag of SOCS3. B, comparison of
SOCS3 binding to different SHP2 phosphotyrosine motifs. Biotinylated
peptides encompassing tyrosine motifs of SHP2 and gp130 were
immobilized on SA chips and the association with SOCS3 was measured by
means of surface plasmon resonance. The concentration of SOCS3 in this
experiment was 15 µM. Nonspecific binding was determined
by using unphosphorylated peptides as well as purified thioredoxin
solution (TRX, 3.5 µM). A phosphorylated
peptide encompassing Tyr-759 of gp130 served as a positive control.
C-F, quantitative comparison of SOCS3-binding to SHP2- and
gp130-peptides. SOCS3 was diluted 2-fold from 34 µM to 17 nM (Y(p)579) or 235 nM (Y(p)542), and the
interaction with the phosphopeptides was measured. Plateau values of
the binding curves were taken for calculation of the
KD values. Graphs used for determination of the
affinity constants for peptides were Y(p)542 (C and
D) and Y(p)759 (E and F). C
and E, sensorgram showing the interaction of serial
dilutions of SOCS3 and the phosphopeptides Y(p)542 and Y(p)759,
respectively. D and F, Scatchard analyses of the
association of SOCS3 with peptides Y(p)542 and Y(p)759, respectively.
Shown is one out of three sets of experiments, which gave similar
results.
2M-promoter induction
by activated chimeric receptors was analyzed (Fig.
7A). Expression of SOCS3
inhibited signal transduction through receptor complexes containing the
cytoplasmic part of wild-type gp130 (IL-5R
/gp130(YYYYYY) · IL-5R
/gp130(YYYYYY)) (compare 1 and 2 in Fig. 7A). In contrast, activation of chimeric receptors
lacking Tyr-759 (IL-5R
/gp130(YFYYYY) · IL-5R
/gp130(YFYYYY)) caused enhanced reporter
activity and is not sensitive to SOCS3 (compare 3 and
4 with 1 and 2). Targeting the C
terminus of wild-type SHP2 to the receptor complex attenuated signal
transduction (compare 6 and 7). The comparison of
7 and 8 shows that SOCS3 hardly affects signal transduction through these receptors.
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Fig. 7.
Targeting of SHP2 to gp130 does not rescue
SOCS3 activity on receptors lacking the SHP2·SOCS3 recruitment site
in gp130. A, HepG2 cells were cotransfected with equal
amounts of expression vectors for IL-5R/gp130 or IL-5R /gp130(YF)/PTP
chimeric receptors. Cells were treated with IL-5 and reporter gene
activity determined as described in Fig. 3B. B,
HepG2 cells were transfected with 2 µg of expression vectors for the
EpoR/gp130 chimeric proteins EG(YYYYYY) or EG(YFYYYY) together with 4 µg of expression vectors for PTP,
-PTP, or the corresponding
control vector and SOCS3 as indicated in the legend together with the
2M-promoter-luciferase reporter construct. Cells were
stimulated for 16 h with 7 units/ml Epo, lysed, and assayed for
luciferase activity as described above.
-PTP) (compare 3 and 4 with 3 and 5 in Fig. 7B). The presence
of membrane-targeted
-PTP did not restore SOCS3 inhibitory activity
on receptors lacking the SOCS3·SHP2 recruitment site (compare
5 and 6). Expression of non-targeted SHP2-PTP
domains (PTP) did not affect signaling through
EpoR/gp130(YFYYYY) as already shown above.
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Fig. 8.
Modulation of Erk activation by SOCS3.
Murine embryonal fibroblasts where transfected with expression vectors
encoding EpoR/gp130(YYYYYY), EpoR/gp130(YFFFFF), or
EpoR/gp130(YYFFFF) and the indicated SOCS3 proteins.
Epo-induced MAPK activity was monitored by using an Elk1
transactivation approach (PathDetect Elk1 trans reporting
system, Stratagene). For the Elk1 reporting system, cells where
transfected with a Gal4-driven luciferase reporter and an
expression vector for a fusion protein composed of the DNA-binding
domain of Gal4 and the transactivation domain of the Elk1 transcription
factor (pFA2-Elk1). Activation of MAPK leads to
phosphorylation/activation of the Elk transactivation domain and
subsequent increase of Gal4/Elk1-dependent luciferase
reporter activity. Thus, modulation of MAPK activity by SOCS3 mutants
can easily be monitored by luciferase activity. Cells were stimulated
with 7 units/ml Epo for 16 h. Transfection efficiency was
monitored by cotransfected -galactosidase expression vector as
described above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Douglas Hilton and Robin Starr for kindly providing SOCS cDNAs.
![]() |
Note Added in Proof |
---|
Bartoe and Nathanson showed that LIF signaling is also independently and negatively regulated by SHP2 and SOCS3 (Bartoe, J. L., and Nathanson, N. M. (2002) Mol. Brain. Res. 107, 108-119).
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FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Fonds der Chemischen Industrie (Frankfurt am Main).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.
§ Both authors contributed equally to this work.
§§ To whom correspondence may be addressed. Tel.: 49-0-241-808-8830; Fax: 49-0-241-808-2428; E-mail: schaper@rwth-aachen.de (F. S.), heinrich{at}rwth-aachen.de (P. C. H.).
Published, JBC Papers in Press, October 27, 2002, DOI 10.1074/jbc.M210552200
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ABBREVIATIONS |
---|
The abbreviations used are: IL-6, interleukin-6; EMSA, electrophoretic mobility shift assay; EPO, erythropoietin; ESS, extended SH2; GAP, GTPase activating protein; gp, glycoprotein; Jak, Janus kinase; KIR, kinase inhibitory region; LIF, leukemia inhibitory factor; MAPK, mitogen-activated protein kinase; OSM, oncostatin M; R, receptor; PTP, protein-tyrosine phosphatase; SH2, Src-homology domain 2; SHP2, SH2 containing protein-tyrosine phosphatase; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; wt, wild-type; Erk1, -2, extracellular signal-regulated kinases 1 and 2; CMV, cytomegalovirus; PP, peptide precipitation.
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REFERENCES |
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