From the INSERM U145, IFR-50, Faculté de Médecine,
06107 Nice Cédex 2, France and the § Walter and Eliza
Hall Institute for Medical Research and the Cooperative Research Center
for Cellular Growth Factor, Parkville, Victoria 3050, Australia
Received for publication, March 12, 2001, and in revised form, April 25, 2001
Suppressor of cytokine signaling (SOCS)
proteins were originally described as cytokine-induced molecules
involved in negative feedback loops. We have shown that SOCS-3 is also
a component of the insulin signaling network (1). Indeed, insulin leads to SOCS-3 expression in 3T3-L1 adipocytes. Once produced, SOCS-3 binds
to phosphorylated tyrosine 960 of the insulin receptor and inhibits
insulin signaling. Now we show that in 3T3-L1 adipocytes and in
transfected COS-7 cells insulin leads to SOCS-3 tyrosine phosphorylation. This phosphorylation takes place on
Tyr204 and is dependent upon a functional
SOCS-3 SH2 domain. Purified insulin receptor directly phosphorylates
SOCS-3. However, in intact cells, a mutant of the insulin receptor,
IRY960F, unable to bind SOCS-3, was as efficient as the wild
type insulin receptor to phosphorylate SOCS-3. Importantly, IRY960F is
as potent as the wild type insulin receptor to activate janus-activated
kinase (Jak) 1 and Jak2. Furthermore, expression of a dominant negative form of Jak2 inhibits insulin-induced SOCS-3 tyrosine phosphorylation. As transfected Jaks have been shown to cause SOCS-3 phosphorylation, we
propose that insulin induces SOCS-3 phosphorylation through Jak
activation. Our data indicate that SOCS-3 belongs to a class of
tyrosine-phosphorylated insulin signaling molecules, the
phosphorylation of which is not dependent upon a direct coupling with
the insulin receptor but relies on the Jaks.
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INTRODUCTION |
Although insulin and cytokines have clearly distinct physiological
functions, some of the molecules they utilize to generate their
cellular responses are shared. For instance, the insulin receptor
substrates (IRS)1 were
originally described as direct and proximal substrates of the insulin
receptor and thought to be specific for the insulin receptor. Later on,
it appeared that these docking proteins are also phosphorylated in
response to several cytokines (2). Conversely, the Stats were
characterized as transcription factors involved specifically in
interferon signaling (3). Several years later, we and others observed
that insulin was capable of inducing the phosphorylation and the
activation of Stat5B (4, 5). Finally, the Jaks are cytosolic tyrosine
kinases activated by cytokines (6), but they were also found to be
stimulated in response to insulin (7, 8).
Recently we proposed that SOCS-3 could be considered as another example
of these shared molecules (1). The SOCS define a family of
proteins with homologous structure, i.e. an
N-terminal region of variable length, a central SH2 domain, and a
C-terminal SOCS box (9-12). Their expression is induced by various
cytokines in a tissue-specific manner. Once expressed, they participate in negative feedback loops, by inhibiting cytokine-mediated Jak/Stat activation. Generally speaking, this inhibition can be mediated by the
following mechanisms: (i) a direct association and inhibition of the
Jak and/or (ii) the association of the SOCS with a
tyrosine-phosphorylated cytokine receptor, leading to a competitive
inhibition with SH2 domain-containing molecules, such as the Stats (13,
14). The physiological importance of this family of proteins was
demonstrated by production of transgenic mice. SOCS-1 deletion causes
perinatal lethality linked to excessive responses to interferon
(15-17). Mice lacking SOCS-2 grow significantly larger than their wild type littermates (18). SOCS-3 knockout results in embryonic lethality
associated with marked erythrocytosis (19).
Originally the SOCS were found to be induced by ligands acting through
receptors belonging to the cytokine receptor family, such as LIF, IL-2,
IL-3, IL-6, interferon
, GH, and leptin (13, 14). We have
shown that insulin induces SOCS-3 expression in 3T3-L1 adipocytes (1).
Moreover, insulin leads to translocation of SOCS-3 from the cytoplasm
to the cell membrane, where it colocalizes with the insulin receptor.
The interaction with the receptor happens through the SH2 domain of
SOCS-3 and the phosphorylated tyrosine 960 of the insulin receptor.
Because Stat5B binds to a domain containing this residue (4), SOCS-3
was found to inhibit insulin-induced Stat5B activation. These data
suggested to us that SOCS-3 was not only a partner of cytokine
signaling but that it also interferes with insulin signal generation.
To further characterize the functional links between the insulin
receptor and SOCS-3 we investigated whether insulin leads to SOCS-3
tyrosine phosphorylation.
We show that insulin induces SOCS-3 tyrosine phosphorylation in 3T3-L1
adipocytes and in transfected COS-7 cells. This phosphorylation takes
place on tyrosine 204 of SOCS-3 and necessitates a functional SOCS-3
SH2 domain. Although in vitro the insulin receptor directly phosphorylates SOCS-3, we found that in intact cells, insulin-induced tyrosine phosphorylation is mediated by the Jaks. Therefore, SOCS-3 belongs to a novel class of distal receptor substrates that are tyrosine-phosphorylated in response to insulin, independently of a
direct coupling with the insulin receptor but dependent on cytosolic
kinases such as the Jaks.
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EXPERIMENTAL PROCEDURES |
Materials--
SOCS-3 antibodies were produced by Eurogentech
(Herstal, Belgium) using a peptide corresponding to the N-terminal
sequence of SOCS-3 (SKFPAAGMSRPLDTSLR). This sequence is specific for
SOCS-3 and is not found in the other known SOCS (9-12). The antibodies were tested for their ability to recognize specifically SOCS-3 by
immunoprecipitation and by Western blotting (data not shown). Antibodies to Jak1, Jak2, and insulin receptor were from Santa Cruz
Biotechnology (Santa Cruz, CA).
Cells, Culture Conditions, and Transfection--
3T3-L1
fibroblasts were obtained from ATCC (Rockville, MD). They were grown
and differentiated into adipocytes as described previously (20). COS-7
fibroblasts were grown in Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) fetal calf serum. 3T3-L1 and COS-7 calls
were starved overnight in Dulbecco's modified Eagle's medium, 0.2%
bovine serum albumin (w/v) before use. COS-7 cells were transfected by
the DEAE-dextran procedure as described (21). Cells were lysed 36 or
48 h after transfection.
Constructs and GST-SOCS-3 Production--
The human insulin
receptor cDNA was obtained from A. Ullrich (Martinsreid, Germany)
and subcloned in pCDNA3. SOCS-3 cDNA, subcloned in pEF, have
been described previously (22). The dominant negative Jak2
8 cloned
in pCDNA3 was described elsewhere (23). Blunt-ended SOCS-3 cDNA
was inserted in-frame with the GST cDNA present in pGex-3X
(Amersham Pharmacia Biotech) blunted after an EcoRI
digestion and filled in by the Klenow fragment. GST and GST-SOCS-3 were
produced according to the manufacturer's instructions (Amersham
Pharmacia Biotech). Point mutations (SOCS-3 Y204F and SOCS-3 R71K) were
produced using the Quickchange site-directed mutagenesis kit from
Stratagene (La Jolla, CA) following the manufacturer's instructions.
All constructions were verified by sequencing.
In Vitro Reconstitution Experiments--
Partially purified
insulin receptors (24) were incubated with insulin (10
7
M) for 45 min at room temperature in (30 mM
Hepes, pH 7.2, 30 mM NaCl, 0.1% (v/v) Triton X-100) and
incubated with GST-SOCS-3 or GST alone adsorbed on Sepharose beads. The
phosphorylation reaction was initiated by adding 15 mM
[
-32P]ATP, 8 mM MnCl2, and 4 mM MgCl2. After 30 min beads were washed twice
in 30 mM Hepes, pH 7.2, 30 mM NaCl, 0.1% (v/v)
Triton X-100. Laemmli buffer was added, and proteins were analyzed by
SDS-PAGE followed by autoradiography.
Preparation of Cell Extracts--
After appropriate treatments
cells were rinsed in ice-cold phosphate-buffered saline and solubilized
in stop buffer (50 mM Hepes, pH 7.2, 150 mM
NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM
Na3VO4, 100 mM NaF, 1% (v/v)
Triton X-100, 10 µg/ml aprotinin, 20 µM leupeptin, 0.18 µg/ml phenylmethylsulfonyl fluoride, 1 mM benzamidine).
SOCS-3 was immunoprecipitated, and the obtained proteins were analyzed by SDS-PAGE under reducing conditions on an 11% polyacrylamide gel.
Proteins were transferred to a polyvinylidene difluoride membrane
(Millipore), and Western blot analyses were performed.
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RESULTS |
Insulin Induces Socs-3 Tyrosine Phosphorylation in COS-7
Cells--
As we have previously shown that SOCS-3 participates in the
insulin signaling network (1), we investigated whether SOCS-3 was
phosphorylated in response to the hormone. COS-7 cells were transiently
transfected with plasmids encoding SOCS-3 and the insulin receptor.
Cells were treated for 5 min with 10
10 to
10
6 M insulin or for 0 to 300 min with
10
7 M insulin. Cells were lysed, and SOCS-3
was immunoprecipitated and analyzed by Western blot using antibody to
phosphotyrosine. In parallel, cell lysates were analyzed for SOCS-3
expression and insulin receptor tyrosine phosphorylation (Fig.
1). As observed, insulin induces an
increase in SOCS-3 tyrosine phosphorylation. This phosphorylation was
detectable after a treatment with 10
9 M of
insulin and reached a maximum at 10
7 M. Thus,
it appears that SOCS-3 phosphorylation occurs at physiologically relevant insulin concentration and with kinetics compatible with insulin action. Phosphorylation was observed after 5 min of insulin treatment and maintained a plateau of phosphorylation for at least 150 min. In all conditions tested SOCS-3 tyrosine phosphorylation paralleled that of the insulin receptor.

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Fig. 1.
Insulin induces SOCS-3 tyrosine
phosphorylation. COS-7 cells were transiently transfected with
plasmids encoding the insulin receptor (1 µg) and SOCS-3 (1 µg) in
100-mm dishes. Cells were treated or not with insulin
(10 6 to 10 10 M) for 5 min or
with 10 7 M insulin for 5 to 300 min. SOCS-3
was immunoprecipitated (IP) and analyzed by
antiphosphotyrosine ( -pTyr) Western blot
(WB). Whole cell lysates (WCL) were analyzed by
antiphosphotyrosine or anti-SOCS-3 Western blot.
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Insulin Receptor Phosphorylates SOCS-3 in Vitro--
We then
tested whether purified insulin receptor phosphorylates SOCS-3. GST or
GST-SOCS-3 were incubated with various amounts of purified insulin
receptors in the presence of a phosphorylation mixture containing
[
-32P]ATP. After 30 min, phosphorylation of GST and
GST-SOCS-3 was analyzed by SDS-PAGE followed by autoradiography (Fig.
2). In the absence of insulin receptor,
GST was phosphorylated by contaminating bacterial kinases. This
contaminating phosphorylation was unaffected by addition of increasing
doses of insulin receptors, indicating that GST is not a substrate of
the insulin receptor. When GST-SOCS-3 was incubated in the presence of
increasing amounts of insulin receptor, its phosphorylation
intensified, indicating that SOCS-3 can be phosphorylated by purified
insulin receptors.

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Fig. 2.
Partially purified insulin receptor
phosphorylates SOCS-3. GST and GST-SOCS-3 adsorbed on Sepharose
beads were incubated in the presence of increasing amounts of purified
insulin receptors and of a phosphorylation mix containing
[ -32P]ATP. After 30 min beads were washed, and
proteins were analyzed by SDS-PAGE. The gel was Coomassie-stained
(right panel) and autoradiographed (left panel).
WGA-IR, wheat germ agglutinin purified insulin
receptor.
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IRY960F Can Induce SOCS-3 Tyrosine Phosphorylation--
Using
yeast two-hybrid experiments we have previously shown that SOCS-3
interacts directly with the phosphotyrosine 960 of the insulin receptor
(1). This view was supported by confocal microscopy illustrating that,
upon insulin stimulation, SOCS-3 and insulin receptor colocalize at the
cell membrane. This interaction is strictly dependent on the
phosphorylation of the insulin receptor tyrosine 960 both in the yeast
two-hybrid system and in intact cells. Therefore, we compared the
ability of a wild type insulin receptor (IRWT) and of the IRY960F
mutant (unable to bind to SOCS-3) to induce SOCS-3 tyrosine
phosphorylation. As a control, we compared the phosphorylation of IRS-1
in cells expressing insulin receptor or IRY960F. COS-7 cells were
transfected with IRWT or IRY960F and with SOCS-3 or with IRS-1. Cells
were treated with insulin and lysed. SOCS-3 or IRS-1 were
immunoprecipitated from the cell lysates and analyzed by
anti-phosphotyrosine Western blot (Fig. 3). Controls for the expression of the
various transfected proteins and for the tyrosine phosphorylation of
the insulin receptors were performed. As previously observed, insulin
induces SOCS-3 tyrosine phosphorylation in cells expressing IRWT.
Unexpectedly, the insulin-induced SOCS-3 phosphorylation was unaltered
in cells expressing IRY960F. In contrast, IRS-1 was less phosphorylated in cells expressing IRY960F compared with cells expressing IRWT, indicating that IRY960F behaves as anticipated for IRS-1 (25). The
remaining level of IRS-1 phosphorylation could be because of endogenous
IGF-I receptor or because of the phosphorylation of IRS-1 by Jak (see
"Discussion"). To summarize, although insulin receptor
phosphorylates SOCS-3 in vitro, and although SOCS-3 binds to
the insulin receptor through phosphotyrosine 960, SOCS-3 does not have
to associate with the insulin receptor to be phosphorylated on
tyrosine.

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Fig. 3.
IRY960F induces SOCS-3 tyrosine
phosphorylation. A, COS-7 cells were transiently
transfected with plasmids encoding IRWT or IRY960F and with SOCS-3
(left panel) or IRS-1 (right panel). Cells were
treated or not with insulin (10 7 M, 5 min).
Whole cell lysates (WCL) were analyzed by
antiphosphotyrosine ( -pTyr) or anti-SOCS-3
(left) or IRS-1 (right) Western blot
(WB). SOCS-3 or IRS-1 were immunoprecipitated
(IP) and analyzed by antiphosphotyrosine Western blot.
IR, insulin receptor.
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IRY960F Induces Jak Phosphorylation--
The Jak kinases appeared
to be likely candidates for this insulin-induced SOCS-3 tyrosine
phosphorylation. Indeed, it has been shown that (i) SOCS-3 interacts
directly with Jak1 and Jak2 through its SH2 domain (26, 27), (ii) these
two cytosolic tyrosine kinases induce SOCS-3 tyrosine phosphorylation
(27), and (iii) insulin activates Jak1 and Jak2 (7, 8, 23). However,
the ability of IRY960F to stimulate the Jak is not established. To
address this issue, we transfected COS-7 cells with IRWT or IRY960F.
Cells were treated or not with insulin, and endogenous Jaks were
immunoprecipitated and analyzed by antiphosphotyrosine Western blot
(Fig. 4). As shown, insulin treatment of
cells expressing IRWT and IRY960F induces tyrosine phosphorylation of
Jak1 and Jak2 to comparable levels. Because it has been reported that
the autophosphorylation of the Jaks in response to insulin parallel their tyrosine kinase activity toward substrates (23), our observations indicate that Jak1 and Jak2 are activated by insulin independently of
the phosphorylation of insulin receptor Tyr960.

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Fig. 4.
IRY960 increases Jak tyrosine
phosphorylation. COS-7 cells were transiently transfected with
plasmids encoding IRWT or IRY960F. Cells were stimulated for 5 min with
insulin (10 7 M). Jak1 and Jak2 were
immunoprecipitated (IP) and analyzed by antiphosphotyrosine
( -pTyr) Western blot (WB).
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Expression of a Dominant Negative Jak Inhibits Insulin-induced
SOCS-3 Phosphorylation--
We then tested the effect of the
expression of a dominant negative form of Jak2 (Jak2
8) on
insulin-induced SOCS-3 tyrosine phosphorylation. This mutant Jak2
8
is a construct mutated within the type VIII phosphotransferase motif of
the C-terminal protein-tyrosine kinase domain and has been described
previously (28). COS-7 cells were transfected with plasmids encoding
insulin receptor, SOCS-3, and various amounts of Jak2
8. Cells were
treated with insulin, and SOCS-3 was immunoprecipitated and analyzed by
antiphosphotyrosine Western blot (Fig.
5). Whole cell lysates were used to
verify insulin receptor tyrosine phosphorylation and SOCS-3 expression. As shown, insulin-induced tyrosine phosphorylation of SOCS-3 was inhibited by increasing amounts of Jak2
8. Jak2
8 did not modify insulin-induced insulin receptor tyrosine phosphorylation or SOCS-3 expression. Together these results suggest that insulin causes SOCS-3 tyrosine phosphorylation through Jak activation.

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Fig. 5.
A dominant negative Jak2 inhibits
insulin-induced SOCS-3 tyrosine phosphorylation. COS-7 cells were
transiently transfected with plasmids encoding the insulin receptor
(IR; 1 µg), SOCS-3 (1 µg), and Jak2 8 (0 to 4 µg) in
100-mm dishes. Cells were treated or not with insulin
(10 7 M) for 5 min. SOCS-3 was
immunoprecipitated (IP) and analyzed by antiphosphotyrosine
( -pTyr) Western blot (WB). Whole
cell lysates (WCL) were analyzed by antiphosphotyrosine or
anti-SOCS-3 ( -SOCS-3) Western blot.
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Insulin-induced SOCS-3 Phosphorylation on Tyrosine 204 Depends on a
Functional SOCS-3 SH2 Domain--
Next we explored in more detail the
insulin-produced SOCS-3 tyrosine phosphorylation. As SOCS-3 binds to
Jak through its SH2 domain, we studied the involvement of SOCS-3 SH2
domain in the insulin-induced SOCS-3 tyrosine phosphorylation. We also
investigated which tyrosine of SOCS-3 is phosphorylated. To do so, we
produced an SH2-defective mutant of SOCS-3 (R71K) in which
Arg71, crucial for the binding of the phosphorylated
tyrosine residue, has been replaced by a Lys. We also prepared several
tyrosine to phenylalanine mutants of SOCS-3, but we will discuss only
Y204F for reasons that will become obvious later on. COS-7 cells were transfected with insulin receptor and with wild type SOCS-3, SOCS-3 Y204F, or SOCS-3 (R71K). Cells were treated or not with insulin, and
SOCS-3 was immunoprecipitated and analyzed by antiphosphotyrosine Western blot (Fig. 6). In parallel, the
quantity of SOCS-3 and the tyrosine phosphorylation of the insulin
receptor were verified using specific antibodies. As previously
observed, insulin induces SOCS-3 tyrosine phosphorylation. When SOCS-3
was mutated on Y204, its tyrosine phosphorylation was nearly
undetectable. With the SOCS-3 (R71K) mutant no phosphorylation was
detected at all. These data indicate that, in response to insulin,
SOCS-3 is phosphorylated on Y204 in a manner dependent on a functional
SOCS-3 SH2 domain.

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Fig. 6.
Insulin-induced phosphorylation of SOCS-3
Tyr204 depends on a functional SOCS-3 SH2 domain.
COS-7 cells were transiently transfected with plasmids encoding the
insulin receptor (1 µg) and SOCS-3, SOCS-3 (Y204F), or SOCS-3
(SH2 ) (1 µg) in 100-mm dishes. Cells were treated or
not with insulin (10 7 M) for 5 min. SOCS-3
was immunoprecipitated (IP) and analyzed by
antiphosphotyrosine ( -pTyr) Western blot
(WB). Whole cell lysates (WCL) were analyzed by
antiphosphotyrosine or anti-SOCS-3 ( -SOCS-3)
Western blot. WT, wild type.
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SOCS-3 Is Phosphorylated on Tyrosine in Response to Insulin in
3T3-L1 Adipocytes--
We used 3T3-L1 adipocytes to determine whether
the hormone induces tyrosine phosphorylation of endogenous SOCS-3. As
SOCS-3 is not expressed in these cells under basal conditions, we
studied its expression after LIF treatment. Differentiated 3T3-L1
adipocytes were treated for 1 to 4 h with LIF. SOCS-3 was
immunoprecipitated and analyzed by Western blot using antibodies to
SOCS-3 (Fig. 7A). SOCS-3 was
not detected in untreated cells, but was induced after 1 h of LIF
treatment. After 2 h the expression decreases and dropped to
non-detectable levels after 4 h. 3T3-L1 adipocytes were
treated for 1 h with LIF and for various times with insulin. SOCS-3 was immunoprecipitated and analyzed by antiphosphotyrosine Western blot (Fig. 7B). A 1-h treatment with LIF induces
SOCS-3 protein expression and its weak tyrosine phosphorylation. This tyrosine phosphorylation was further increased by insulin. It was
observed as soon as 2.5 min after treatment and appeared to reach a
plateau within 7.5 min. We next investigated whether insulin-produced SOCS-3 tyrosine phosphorylation was specific for SOCS-3 induced by LIF.
3T3L1 adipocytes were treated for 2, 4, and 6 h with insulin alone
or pretreated for 1 h with LIF or GH and then stimulated with
insulin. SOCS-3 was imunoprecipitated and analyzed by
antiphosphotyrosine or by anti-SOCS-3 Western blot (Fig.
7C). Treatment with LIF and GH induced comparable levels of
SOCS-3 expression. Insulin leads to SOCS-3 expression after 2 and
4 h of treatment, but after 6 h the expression becomes nearly
undetectable. LIF and GH alone produce a weak increase in SOCS-3
tyrosine phosphorylation. In both conditions this basal phosphorylation
is further increased after insulin treatment. Exposure of cells to
insulin alone leads also to SOCS-3 tyrosine phosphorylation. This
phosphorylation reached a maximum within 2 h of treatment and
decreased after 4 h, although SOCS-3 expression was maintained at
this time. We conclude that in 3T3-L1 adipocytes endogenous SOCS-3 is
tyrosine-phosphorylated in response to insulin.

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Fig. 7.
Insulin increases SOCS-3 tyrosine
phosphorylation in 3T3-L1 adipocytes. A, 3T3-L1
adipocytes were treated with LIF (10 nM) for 0 to 4 h.
SOCS-3 was immunoprecipitated (IP) and analyzed by
anti-SOCS-3 ( -SOCS-3) Western blot
(WB). B, 3T3-L1 adipocytes were treated for
1 h with Na3V2O4 (200 µM) and then with or without LIF (10 nM) for
1 additional hour to induce SOCS-3 expression. Cells previously treated
with LIF were then stimulated with or without insulin for 2.5, 5, 7.5, or 10 min. SOCS-3 was immunoprecipitated and analyzed by Western blot
with antibodies to phosphotyrosine ( -pTyr).
C, 3T3-L1 adipocytes were pretreated for 1 h with
Na3V2O4 (200 µM).
Cells were then treated for 1 h with LIF (10 nM) or GH
(96 nM) or with insulin (10 7 M)
for 120, 240, or 360 min. Cells pretreated with LIF or GH were then
stimulated or not with insulin (10 7 M) for 10 min. Cells were lysed, and SOCS-3 was immunoprecipitated and analyzed
by antiphosphotyrosine Western blot. The membrane was stripped and
probed for SOCS-3 expression.
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DISCUSSION |
We have previously presented evidence for the idea that SOCS-3 is
a component of the insulin signaling network (1). Indeed, insulin
induces SOCS-3 mRNA expression in 3T3-L1 adipocytes. In addition,
insulin leads to the translocation of SOCS-3 from the cytoplasm to
the plasma membrane where it colocalizes with the insulin receptor.
This interaction with the receptor could be a mean for desensitization
of insulin signaling, because we have shown that SOCS-3 inhibits
insulin-dependent Stat5B activation. Here we show that
insulin leads to SOCS-3 tyrosine phosphorylation in transfected COS-7
cells and in 3T3-L1 adipocytes after induction of endogenous SOCS-3.
Interestingly, it would appear that in intact cells SOCS-3 does not
have to be coupled directly to the insulin receptor to be
phosphorylated on tyrosine. This is in apparent contradiction with our
observation that purified insulin receptor phosphorylates SOCS-3 and
that SOCS-3 binds to the insulin receptor phosphotyrosine 960. Indeed,
using the yeast two-hybrid system, we have shown that SOCS-3 binds
through its SH2 domain to the phosphotyrosine 960 of the insulin
receptor (1). Further, in cells expressing a mutant of the receptor
(IRY960F), which does not bind SOCS-3, insulin does not modify the
cytoplasmic localization of SOCS-3. However, insulin-induced SOCS-3
tyrosine phosphorylation in cells expressing IRY960F is comparable with
that seen in cells expressing wild type insulin receptor (Fig. 3). This
observation is intriguing, because most recognized substrates of the
insulin receptor, such as the IRS, Shc, and Gab, have to be coupled to the insulin receptor to be phosphorylated on tyrosine (29, 30). This
interaction can be direct, through the phosphotyrosine binding domain of the IRS and Shc to tyrosine 960 of the insulin receptor or
through the IRS-2 kinase regulatory binding domain to the
so-called regulatory loop domain of insulin receptor (31). The coupling can also be indirect through the plekstrin homology domain of IRS and Gab-1 (32, 33). These plekstrin homology domains may bind
acidic peptide motifs in membrane proteins or phospholipids that link
substrates to activated cell surface insulin receptors (34).
Differently from these proximal substrates, SOCS-3 does not require to
be localized to the cell membrane and coupled with the insulin receptor
to be phosphorylated on tyrosine in response to insulin. Therefore, it
is likely that in intact cells, the insulin receptor does not directly
phosphorylate SOCS-3 but utilizes an intermediary tyrosine kinase for
this to be achieved. Several experimental observations indicate that
the Jaks are the kinases involved. First, it has already been reported
that the Jaks are activated in response to insulin. This was observed
originally in cells overexpressing insulin receptors (7) and later
confirmed in intact rats where injection of insulin stimulates Jak2 in
liver, muscle, and adipose tissue (8). This activation is probably linked to a coupling of the Jak to the insulin receptor. Indeed, using
GST fusion proteins and coimmunoprecipitation experiments, our
laboratory showed that the Jaks interact directly with the insulin
receptor (23). This interaction involves two domains of Jak1, one
located in its N terminus and the other located in its C terminus, and
necessitates a phosphorylated insulin receptor. Second, it has also
been already observed that ectopic expression of Jak1 or Jak2 induces
SOCS-3 tyrosine phosphorylation (27). This can be correlated with the
direct association between the Jak and SOCS-3 (26, 27). Interestingly,
we show that a mutant of SOCS-3 defective in its SH2 domain is not
phosphorylated in response to insulin, probably because (as previously
reported) it can not bind to Jak (26). Third, both wild type insulin
receptor and IRY960F cause a comparable phosphorylation of Jak1 and
Jak2, which is in agreement with the ability of these two receptors to
induce SOCS-3 tyrosine phosphorylation. Fourth, the use of a dominant
negative Jak2 mutant showed that the Jaks are necessary to mediate
insulin-induced tyrosine phosphorylation of SOCS-3 (Fig. 5). Such
kinase-dead Jak2 mutants have previously been reported to inhibit the
effect of both Jak1 and Jak2 on Stat3 activation by IGF-I, for example
(35). Therefore, we would like to suggest that insulin stimulates the
Jaks in a manner independent of the phosphorylation of insulin receptor
tyrosine 960. Once activated, some of the Jak molecules would
translocate to the cytoplasm, capture the SOCS-3 through binding to
their SH2 domain, and then phosphorylate the SOCS-3.
Schematically speaking, it would appear that SOCS-3 can interfere at
different levels in insulin signaling. Indeed, we found earlier that
SOCS-3 inhibits insulin-induced Stat5 activation probably by competing
for the same phosphotyrosine 960 on the receptor (1). Here we show that
insulin leads to SOCS-3 tyrosine phosphorylation. It is likely that
this phosphorylation has an impact on insulin signaling. We did not
detect a striking difference in the subcellular localization of SOCS-3
and SOCS-3 Y204F, before or after insulin treatment, and both molecules
inhibit insulin-induced Stat-5B activation in a similar manner (data
not shown). However, SOCS-3 Tyr204 is located in close
vicinity to the consensus elonginBC (the complex of elongin B
and elongin C) binding site (36, 37). Because the function of the
association between SOCS-3 and eloginBC (the complex of elongin B and
elongin C) is still a matter of debate, it is difficult to predict the
effect of SOCS-3 tyrosine phosphorylation on this event (36, 37). It
can also be envisioned that SOCS-3 tyrosine phosphorylation may create
binding sites for SH2-containing molecules. SOCS-3 would be then able
to create a bridge between the insulin receptor and a SH2-containing
molecules. This would be reminiscent of Shp-2, which binds to the
platelet-derived growth factor receptor through its SH2 domain and to
Grb2 through its phosphorylated tyrosine (38). This docking function of
SOCS-3 could be occurring when SOCS-3 is in the cytosol. After its
translocation to the cell surface SOCS-3 could function as a cargo
bringing proteins in vicinity to the insulin receptor. SOCS-3, located at the cell surface, could modify the functioning of the insulin receptor by docking proteins.
Our observations add weight to the role of the Jaks in insulin
signaling. Indeed, although the Jaks have been shown to induce IRS-1
and IRS-2 tyrosine phosphorylation, their relative contribution to this
process, compared with the direct phosphorylation of IRS by the insulin
receptor, is thought to be small. This view comes from the finding that
mutation of the insulin receptor on tyrosine 960 leads to a decrease in
insulin-induced IRS-1 tyrosine phosphorylation (25). However, it can be
noted that as shown in Fig. 3 (and as observed by other authors) some
insulin-induced IRS-1 phosphorylation is still detectable in cells
expressing IRY960F (39, 40). This remaining phosphorylation could be
because of the Jaks. Because we have shown earlier that the
phosphopeptide maps of IRS-1 phosphorylated in vitro by
insulin receptor versus Jak1 are different (23), it is
tempting to think that the docking function of IRS-1 will also differ
and consequently so will its signaling potential.
In summary, to the best of our knowledge, SOCS-3 appears as the first
example of a protein tyrosine phosphorylated in intact cells in
response to insulin in a fashion that is entirely
Jak-dependent. However, it is possible that other proteins
share this property. Several substrates of the insulin receptor (such
as Tub; see Ref. 41) have been proposed to be direct substrates of the
insulin receptor based on their phosphorylation by purified insulin
receptors. Considering our present work the possible implication of the
Jaks should be evaluated. Finally, it is tempting to suggest that
insulin uses the Jak activity not only to modulate its action through SOCS-3 but also to generate specific biological responses.
The abbreviations used are:
IRS, insulin
receptor substrate;
Stat, signal transducer and activator of
transcription;
SOCS, suppressor of cytokine signaling;
LIF, leukemia
inhibitory factor;
Jak, janus-activated kinase;
IL, interleukin;
GH, growth hormone;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
IRWT, wild type insulin
receptor.
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