IL-11 (
)is a stromal fibroblast-derived cytokine (1, 2) that plays essential roles in a variety of
biological systems. Previous studies have suggested that IL-11 depends
on gp130 to transduce its signals into cells(3) . gp130 is an
IL-6 receptor-associated signal transducer and is shared by receptor
complexes for leukemia inhibitor factor, oncostatin M, ciliary
neurotrophic factor, and IL-11(3) . It has been reported that
gp130 associates with Janus kinase 2 (JAK2) tyrosine kinase, which is
tyrosine-phosphorylated and activated upon IL-11, IL-6, ciliary
neurotrophic factor, oncostatin M, or leukemia inhibitor factor
stimulation of cells(4, 5) . The response of JAK2 to
IL-6 is blocked by point mutations or deletions of the membrane
proximal portion of gp130 (6) . It was further demonstrated
that mitogen-activated protein kinase (MAPK) is tyrosine-phosphorylated
and activated following IL-11 stimulation(7) . MAPK has been
shown to be a key target of Ras(8) , a crucial downstream
signaling partner for growth factor receptor tyrosine
kinases(9, 10) . Thus, it is possible that Ras may
also be involved in IL-11 signal transduction.
Growth factor
receptor binding protein 2 (Grb2) is an adaptor protein containing a
Src homology type 2 (SH2) domain flanked by two Src homology type 3
(SH3) domains(9, 10, 11) . Through its SH3
domains, Grb2 constitutively binds a Ras guanine nucleotide-releasing
factor (GNRF) like Sos or Cdc25, which activates Ras by replacing
Ras-bound GDP with GTP. In the absence of cellular activation, the
Grb2
GNRF complex is located in the cytoplasm. Upon activation by
growth factors, the SH2 domain of Grb2 binds activated
tyrosine-phosphorylated receptors or receptor-associated tyrosine
phosphoproteins, thus bringing GNRF to the plasma membrane where Ras is
located(9, 10, 11) . Through its adaptor-like
function, Grb2 plays a key role in linking cell receptor tyrosine
kinases and associated tyrosine phosphoproteins with the Ras pathway.
The interactions of Grb2 with receptor tyrosine kinases have been
studied in the epidermal growth factor receptor (EGFR) and the insulin
receptor (IR) systems(12, 13) . Ligand binding leads
to EGFR autophosphorylation on multiple tyrosine residues.
Subsequently, the SH2 domain of Grb2 becomes attached to tyrosine
residues of the EGFR, thus relocating Sos to the plasma membrane. In
contrast to the EGFR, stimulation of the IR involves two additional
proteins, Shc and insulin receptor substrate-1. Upon stimulation with
insulin, Shc and insulin receptor substrate-1 become
tyrosine-phosphorylated and bind to Grb2 in order to recruit the
Grb2
Sos complex to the cell membrane.
In marked contrast to
receptor tyrosine kinases like EGFR and IR, the IL-11 signal transducer
gp130 has no kinase domains (3) and needs to activate
membrane-associated non-receptor tyrosine kinases for mediating its
effects. It is therefore interesting to elucidate which non-receptor
tyrosine kinases are recruited by IL-11 to interact with Grb2, hence
transducing signals from the activated IL-11 receptor
gp130
complex to the Ras/MAPK pathway.
EXPERIMENTAL PROCEDURES
Materials
Recombinant human IL-11 (specific
activity, 2.5
10
units/mg) was generously provided
by Genetics Institute (Cambridge, MA). Anti-phosphotyrosine monoclonal
antibody (4G10), and affinity-purified anti-gp130 and anti-JAK2
antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY).
Affinity-purified anti-Grb2 and anti-Fyn antibodies, together with
anti-Fyn monoclonal antibody were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Anti-Ras monoclonal antibody (Y13-259) was from
Oncogene Science, Inc. (Cambridge, MA). Anti-Syp was a gift from Dr.
Gen-Sheng Feng (Indiana University School of Medicine). Horseradish
peroxidase-conjugated anti-mouse and anti-rabbit IgG were from Amersham
Corp., and horseradish peroxidase-conjugated anti-rat IgG was from
Zymed Laboratories, Inc. (San Francisco, CA).
Treatment of 3T3-L1 Cells with IL-11,
Immunoprecipitation, and Immunoblotting
3T3L1 mouse
preadipocytes were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mML-glutamine. Confluent cells were stimulated without or
with 500 ng/ml IL-11 at 37 °C as indicated under ``Results and
Discussion.'' 500 ng/ml has been determined to be the optimum
concentration of IL-11 on 3T3L1 cells from our previous
studies(7) . Reactions were stopped by placing dishes of
attached cells on ice and adding ice-cold phosphate-buffered saline
containing 2 mM sodium orthovanadate to the cells. Cells were
scraped off the plate, washed in ice-cold phosphate-buffered saline,
and then centrifuged at 500
g for 5 min at 4 °C.
The cells were then lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1%
Nonidet P-40, 0.25% sodium deoxycholate, 1 mM
phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM sodium
orthovanadate, 1 µg/ml aprotinin, and 1 µg/ml leupeptin).
Solubilized proteins were collected after centrifugation (13,000
g for 10 min at 4 °C) and used for
immunoprecipitations with the appropriate antibodies for 4-h incubation
at 4 °C with rotation. The immune complexes were further incubated
with protein A-agarose beads (Upstate Biotechnology, Inc., Lake Placid,
NY) for 1 h at 4 °C with rotation and then washed five times with
modified RIPA buffer. The immunoprecipitates were separated by 12 or
7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to
polyvinylidine difluoride membranes. The membranes were then
immunoblotted with the appropriate antibodies, and antibody-bound
proteins were visualized using horseradish peroxidase-conjugated
anti-mouse, anti-rabbit, or anti-rat IgG and ECL (Amersham Corp.)
according to the manufacturer's specifications.
In Vitro Binding Assay
GST-Raf (amino acids
51-149) fusion protein and GST alone as a control were freshly
prepared for in vitro binding experiments as
described(14) . 3T3L1 cells were treated in the absence or
presence of 500 ng/ml IL-11 at 37 °C as indicated under
``Results and Discussion.'' For in vitro binding
studies, glutathione-Sepharose beads (Pharmacia Biotech Inc.) with
bound fusion proteins (approximately 5 µg of fusion protein/binding
reaction) were incubated with cell lysates in digitonin lysis buffer
(1% digitonin, 150 mM NaCl, 2 mM EDTA, 50 mM Tris, pH 8.0, 2 mM sodium orthovanadate, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin) at 4 °C for 12-14 h.
The beads were washed three times with digitonin lysis buffer before
the bound proteins were separated by SDS-PAGE and immunoblotted with
anti-Ras.
RESULTS AND DISCUSSION
IL-11 Induces the GTP-Ras Formation in 3T3L1
Cells
In order to test whether Ras was activated by IL-11, the
GST-Raf in vitro binding assay was performed using lysates
from unstimulated and IL-11-treated 3T3L1 cells. It has recently been
shown that Ras protein interacts directly with GST-Raf in
vitro, and the binding of Ras and GST-Raf is dependent on the
activation of Ras loaded with GTP rather than GDP(14) .
Therefore, the in vitro binding assay of Ras and GST-Raf is
suggested to be a useful approach to verify the activation of
Ras(14) . In this study, we demonstrated that IL-11 induces
complex formation between Ras and GST-Raf (amino acids 51-149) in vitro (Fig. 1). 3T3L1 cells were treated without or
with 500 ng/ml IL-11 for 3 min and lysed with digitonin-containing
lysis buffer. Cell lysates were immunoprecipitated with anti-Ras
(Y13-259) or incubated with GST-Raf fusion protein or GST alone
(as a negative control) immobilized to glutathione-Sepharose. The
samples were washed, and bound proteins were separated by SDS-PAGE and
immunoblotted with anti-Ras Ab (Y13-259). As shown in Fig. 1, stimulation of 3T3L1 cells with IL-11 had no effect on
the protein level of Ras as was determined by immunoprecipitation with
anti-Ras. Ras complexed with GST-Raf fusion proteins in the lysates
from IL-11-stimulated 3T3L1 cells, whereas no GST-Raf-associated Ras
was detected from unstimulated 3T3L1 cells. These results in
conjunction with our previous studies (7) indicate that IL-11
signaling is at least in part mediated through the Ras/MAPK pathway.
Figure 1:
IL-11
induces the association of Ras with GST-Raf in vitro. 3T3L1
cells were untreated or stimulated with IL-11 (500 ng/ml) for 3 min.
Cell lysates were incubated with anti-Ras mAbs (Y13-238), GST-Raf
fusion protein, or GST protein. The bound proteins were separated by
12% SDS-PAGE and immunoblotted with anti-Ras mAbs
(Y13-259).
Phosphotyrosine-containing Proteins Associate with Grb2
in 3T3L1 Cells
The adaptor protein Grb2 is a key intermediate to
facilitate protein-protein interactions and to transduce signals from
ligand-activated membrane receptors to the Ras/MAPK pathway. To analyze
the pattern of phosphotyrosine-containing proteins associated with Grb2
following IL-11 stimulation, lysates in modified RIPA buffer from
IL-11-treated or untreated 3T3L1 cells were immunoprecipitated with
anti-Grb2 antibody, and the precipitated proteins were analyzed by
immunoblotting with anti-phosphotyrosine mAbs (4G10). As shown in Fig. 2, Grb2 constitutively associated with a tyrosine
phosphoprotein with a molecular mass around 75 kDa, and IL-11
stimulation did not seem to affect their association. However, IL-11
induced two more phosphotyrosine-containing proteins of 130 and 60 kDa
to complex with Grb2. After IL-11 treatment, the association of these
two phosphotyrosine-containing proteins with Grb2 occurred at 1 min,
could still be observed at 3 min, and completely disappeared at 7 min.
1 and 7 min are selected to be the two time points of IL-11 treatment
for subsequent experiments. The same immunoprecipitates were reblotted
with anti-Grb2 to verify the same amount of Grb2 in each
immunoprecipitate. The 75-kDa tyrosine phosphoprotein was further
identified as Syp (data not shown). Syp is a newly discovered
protein-tyrosine phosphatase(15) , and the function of its
association with Grb2 in IL-11-triggered signal transduction requires
further investigation.
Figure 2:
Grb2 associates with
phosphotyrosine-containing proteins. 3T3L1 cells were untreated or
stimulated by IL-11 (500 ng/ml) for the indicated times. Cell lysates
were immunoprecipitated with anti-Grb2. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted by
anti-phosphotyrosine mAb (4G10) and
anti-Grb2.
Grb2 Associates with Tyrosine-phosphorylated Fyn in
Response to IL-11
To identify the 60-kDa Grb2-associated
tyrosine-phosphorylated protein, anti-Grb2 immunoprecipitates were
immunoblotted with anti-Shc. Shc has been shown to complex with Grb2
and activate the Ras signaling pathway upon stimulation with several
cytokines(16, 17) . However, in our experiments Shc
was not detected in anti-Grb2 immunoprecipitates from both unstimulated
and IL-11-treated 3T3-L1 cells (data not shown). We therefore examined
whether Src family tyrosine kinases, including Fyn, Yes, and Src, are
possible candidates for this 60-kDa tyrosine-phosphorylated protein.
Fyn, Yes, and Src have been determined from our previous studies to be
the three Src family kinases expressed in 3T3L1 cells (data not shown).
As shown in Fig. 3A, Fyn was not present in anti-Grb2
immunoprecipitates from unstimulated cells. It appeared in anti-Grb2
immunoprecipitates after cells were stimulated with IL-11 for 1 min and
disappeared after cells were exposed to IL-11 for 7 min. Src and Yes
could not be detected in anti-Grb2 immunoprecipitates from either
unstimulated or IL-11-treated 3T3L1 cells (data not shown). The
reciprocal immunoprecipitation and immunoblotting confirmed the
presence of Grb2 in the anti-Fyn immunoprecipitates after cells were
stimulated by IL-11 for 1 min, as shown in Fig. 3B. Fyn
has a basal level of tyrosine phosphorylation in the unstimulated
3T3-L1 cells. However, tyrosine phosphorylation of Fyn was enhanced
transiently following IL-11 stimulation (Fig. 3B). Fyn
and other Src family kinases have been reported to be associated with
signaling molecules, including phospholipase C-
2 and PI3-kinase,
which phosphorylates the Asp-3 position of phosphatidylinositol in
various cytokine signal transduction
mechanisms(18, 19, 20) . It has also been
reported that Src family kinases are involved in the regulation of Ras
activity. The Ras GTP-activating protein, an important regulator of the
Ras function, was found in complexes with v-Src and c-Src and was
phosphorylated by activated Src kinase in v-Src- and c-Src-transformed
cells(21) . In NIH3T3 fibroblasts expressing oncogenic and
transformation-defective variants of the Src family kinase
p56
, tyrosine phosphorylation and activation of Ras
GTP-activating protein required the enzymatic activation and
myristoylation of p56
(22) . Recently, the
p120
protooncogene product has been shown to form a
complex with Fyn, Grb2, and PI3-kinase(23) . The association of
p120
with Fyn and PI3-kinase was markedly increased by T
cell activation. In contrast, the association of p120
with Grb2 did not change upon T cell activation. Our present
results further suggest that Fyn can be recruited to interact with Grb2
following IL-11 stimulation, thus taking a part in transducing signals
from the IL-11 receptor
gp130 complex to the Ras pathway.
Figure 3:
Tyrosine-phosphorylated Fyn associates
with Grb2 in response to IL-11. 3T3L1 cells were treated without or
with IL-11 (500 ng/ml) for the indicated times. Cell lysates were
immunoprecipitated with either anti-Grb2 (A) or anti-Fyn (B). The immunoprecipitates (IP) were resolved by
SDS-PAGE and sequentially immunoblotted with anti-Fyn and anti-Grb2 (A) or anti-Grb2, anti-phosphotyrosine mAb (4G10), and
anti-Fyn (B).
IL-11 Induces Tyrosine-phosphorylated JAK2 to Complex
with Grb2 and Fyn as well as gp130
The 130-kDa Grb2-bound
tyrosine-phosphorylated protein has a similar molecular weight as the
JAK family tyrosine kinases. To test whether Grb2 binds JAK family
tyrosine kinases, cell lysates were immunoprecipitated with either
anti-Grb2 or anti-JAK2 antibodies, and these immune complexes were then
analyzed by immunoblotting with anti-JAK2 and anti-Grb2. JAK2 can be
detected in anti-Grb2 immunoprecipitates only after cells were
stimulated by IL-11 for 1 min but not from control cells or cells that
were exposed to IL-11 for 7 min (Fig. 4A). Related to
this experiment, Grb2 was present in anti-JAK2 immunoprecipitates only
after cells were treated by IL-11 for 1 min (Fig. 4B).
JAK1 was not detected in anti-Grb2 immunoprecipitates from both
unstimulated and IL-11-treated 3T3-L1 cells (data not shown). Fig. 4B also shows that tyrosine phosphorylation of
JAK2 is transiently induced after IL-11 treatment. Fyn can also be
found in anti-JAK2 immunoprecipitates after 3T3L1 cells were stimulated
by IL-11 (Fig. 4B). The results demonstrate that IL-11
induces Grb2 and Fyn to complex with tyrosine-phosphorylated JAK2. JAK
kinase activation may result in tyrosine phosphorylation and activation
of the Stat family transcription factors, which were originally
described in the interferon system(24) . Previous studies have
shown that JAK2 associates with growth hormone receptor and
erythropoietin receptors and is activated by the ligand receptor
binding(25, 26) . It has been reported (4) and
is confirmed in this paper that JAK2 associates with IL-11 signal
transducer gp130 (Fig. 4C). By association with gp130
and Grb2, JAK2 appears to play a critical role in transducing signals
from the activated IL-11 receptor
gp130 complex to the Ras/MAPK
pathway via Grb2. Our experiments further showed that JAK2 associates
with Fyn after IL-11 treatment (Fig. 4B). It has been
theorized that protein-protein interactions may cause a conformational
change in the Src family kinases, thus leading to the activation of the
Src family kinases(27) . JAK2 has also been found to form a
complex containing Src and PI3-kinase in response to IL-3 in NIH3T3
cells(28) . It is therefore possible that JAK2 may associate
with and hence activate Fyn after IL-11 stimulation.
Figure 4:
IL-11 induces tyrosine-phosphorylated JAK2
to complex with Grb2 and Fyn as well as gp130. 3T3L1 cells were treated
without or with IL-11 (500 ng/ml) for the indicated times. Cell lysates
were immunoprecipitated with anti-Grb2 (A), anti-JAK2 (B), or anti-gp130 (C). The immunoprecipitates (IP) were resolved by SDS-PAGE and sequentially immunoblotted
with anti-JAK2 and anti-Grb2 (A), anti-Grb2, anti-Fyn (mAb),
anti-phosphotyrosine mAb (4G10), and anti-JAK2 (B), or
anti-JAK2 and anti-gp130 (C).
In summary, our
data demonstrate that IL-11 stimulation induces a novel complex
formation among JAK2, Fyn, and Grb2. The complex may serve to propagate
signals originating from the IL-11 receptor
gp130 or alter the
subcellular location of key signaling molecules such as Grb2-bound Ras
GNRF. Grb2-complexed GNRFs identified so far are Sos1(29) ,
Sos2 (29) , Cdc25(30) , and C3G(31) . Our
preliminary experiments failed to demonstrate the association between
mSos1 and JAK2 or Fyn (data not shown). Further investigations will be
required to identify the Grb2-bound GNRF, which is involved in
IL-11-induced Ras activation. In addition to providing a novel
mechanism that activates the Ras/MAPK signaling pathway, IL-11-induced
complex formation of Grb2, JAK2, and Fyn may also represent the
interactions among JAK/Stat, Ras/MAPK, and other Src family
kinase-related signaling pathways. These interactions may result in
diverse signaling pathways functioning in concert to dictate different
cellular responses.