From INSERM U151, Institut Louis Bugnard, IFR 31, CHU Rangueil, 31403 Toulouse Cedex 04, France and § Institut Cochin de Génétique Moléculaire, 75014 Paris, France
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ABSTRACT |
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We have previously reported in Chinese hamster ovary (CHO) cells expressing sst2 that activation of the sst2 somatostatin receptor inhibits insulin-induced cell proliferation by a mechanism involving stimulation of a tyrosine phosphatase activity. Here we show that the tyrosine phosphatase SHP-1 was associated with the insulin receptor (IR) at the basal level. Activation of IR by insulin resulted in a rapid and transient increase of tyrosine phosphorylation of IR, its substrates IRS-1 and Shc, and also of SHP-1. This was then followed by a rapid dephosphorylation of these molecules, which was related to the insulin-induced increase of SHP-1 association to IR and of SHP-1 activity. On the other hand, addition to insulin of the somatostatin analogue, RC160, resulted in a higher and faster increase of SHP-1 association to IR directly correlated with an inhibition of phosphorylation of IR and its substrates, IRS-1 and Shc. RC160 also induced a higher and more sustained increase in SHP-1 activity. Furthermore, RC160 completely suppressed the effect of insulin on SHP-1 phosphorylation. Finally, in CHO cells coexpressing sst2 and a catalytically inactive mutant SHP-1, insulin as well as RC160 could no longer stimulate SHP-1 activity. Overexpression of the SHP-1 mutant prevented the insulin-induced signaling to be terminated by dephosphorylation of IR, suppressed the inhibitory effect of RC160 on insulin-induced IR phosphorylation, and abolished the cell proliferation modulation by insulin and RC160. Our results suggest that SHP-1 plays a role in negatively modulating insulin signaling by association with IR. Furthermore, somatostatin inhibits the insulin-induced mitogenic signal by accelerating and amplifying the effect of SHP-1 on the termination of the insulin signaling pathway.
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INTRODUCTION |
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Somatostatin is a neuropeptide localized in numerous mammalian tissues. It exerts pleiotropic biological processes including neurotransmission, inhibition of hormonal and hydroelectrolytic secretions, and cell proliferation. This neuropeptide induces its biological effects by interacting with specific receptors that belong to the seven-transmembrane domain receptor superfamily. Five receptors have been recently cloned (sst1-5) and shown to mediate a variety of signal transduction pathways as inhibition of adenylate cyclase and guanylate cyclase, modulation of ionic conductance channels, and protein dephosphorylation (1-4). The five receptors bind to the natural peptides, somatostatin 14 and somatostatin 28, with high affinity, whereas somatostatin analogues selectively interact with sst2, sst3 and, sst5 (4-8).
Somatostatin and its stable analogues promote growth inhibition of various normal and tumor cells (9, 10). In pancreatic tumors cells, we demonstrated that somatostatin and analogues antagonize the mitogenic effect of growth factors acting on tyrosine kinase receptors such as epidermal growth factor (11). Somatostatin peptides also cause a rapid stimulation of a membrane protein tyrosine phosphatase (PTPase)1 activity and dephosphorylate epidermal growth factor receptors (12, 13). Among the five somatostatin receptors, it has recently been shown that sst2 selectively mediates the inhibitory effect of somatostatin analogues on serum- or insulin-induced cell proliferation by a mechanism involving stimulation of a PTPase in NIH 3T3 and Chinese hamster ovary (CHO) cells expressing sst2 (14, 15). We further demonstrated that a PTPase, identified as SHP-1, co-purified with somatostatin receptors in pancreatic acinar cells that highly expressed sst2 (16). Recent studies have revealed that SHP-1 associates with sst2 and becomes activated in response to somatostatin, suggesting that SHP-1 may be part of the antiproliferative signal promoted by sst2 (17).
SHP-1, a Src homology 2 (SH2) domain containing intracellular PTPase, is predominantly expressed in multiple hematopoietic lineages and to a lesser degree in epithelial cells (18-21). This enzyme has been described to interact with numerous activated growth factor, cytokine, and antigen receptors. It is also implicated in the negative regulation of various immunoreceptor transduction pathways by dephosphorylating and inactivating these receptors or their cognate substrates (22-25). The essential role of SHP-1 as a negative regulator of hematopoietic cell signal transduction is consistent with the multiple defects in hematopoietic cells observed in motheaten mice characterized by mutations in the SHP-1 gene and loss of SHP-1 activity (26).
The identification of substrates of SHP-1 is essential for understanding the negative growth signal promoted by sst2. Because activated sst2 inhibits the mitogenic signal promoted by insulin, the insulin receptor is a potential candidate. Thus, we analyzed the early steps occurring downstream of activation of insulin receptor and sst2 in CHO cells expressing sst2. Our data suggest that SHP-1 is one negative regulator of the insulin signaling, and that upon activation, sst2 negatively modulates insulin signaling by accelerating and amplifying the regulatory functions of SHP-1.
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MATERIALS AND METHODS |
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Reagents--
Monoclonal anti-SHP-1 and anti-Shc antibodies and
polyclonal anti-IR (
subunit of insulin receptor (IR)) antibodies
were purchased from Transduction Laboratories. Anti-IRS-1 antibodies were from Upstate Biotechnology Inc. Monoclonal anti-phosphotyrosine (PY-20) antibodies were from Santa-Cruz Biotechnology. RC-160 was a
kind gift from Dr A. V. Schally (Tulane University, New Orleans,
LA).
Construction and Expression of the sst2-SHP-1 Mutant in CHO Cells-- The 1.2-kilobase XbaI fragment of mouse sst2A cDNA subcloned into pCMV6c vector was stably co-transfected in CHO (DG44 variant) cells using Lipofectin reagent with pSV2neo as described previously (Dr. G. I. Bell, Howard Hughes Medical Institute, University of Chicago, and Dr. T. Reisine, University of Pennsylvania, School of Medicine, Philadelphia) (4). Stable transfectants were selected in Dulbecco's modified Eagle's medium containing geneticin at 600 µg/ml. Geneticin-resistant clones expressing sst2 (CHO/sst2) were screened for somatostatin binding using [125I-Tyr11]somatostatin as tracer as described previously (14). The 2.1-kilobase HindIII/NotI fragment of human SHP-1 cDNA (Dr. M. L. Thomas, Howard Hughes Medical Institute, Washington University, St Louis, MO) was subcloned into the expression vector pcDNA I neo vector (Invitrogen). The SHP-1(C453S) mutant was constructed with the oligonucleotide primer 5'-GAT GCC AGC GCT GGA ATG CAC AAT-3' by using the method of Kunkel et al. (27). The mutation was confirmed by dideoxynucleotide sequencing. The mouse sst2 gene in the pCMV6c vector was stably co-transfected in CHO cells using Lipofectin reagent with the SHP-1(C453S) mutant in pcDNA I neo. Stable colonies obtained by selection with G418 (600 µg/ml) were screened for somatostatin binding and cellular clones expressing somatostatin binding sites at similar levels to CHO/sst2 clones were screened for the presence of SHP-1 using Western blot analysis as described below.
Cell Culture and Growth Assay--
CHO-DG44 stably expressing
the cloned mouse sst2 (CHO/sst2) or sst2 and SHP-1(C453S)
(CHO/sst2-SHP-1(C453S)) were cultured in -minimal essential medium
(
MEM) containing 10% fetal calf serum and G418 (200 µg/ml) as
described previously (14). For cell treatment, cells were plated in
100-mm diameter dishes (75 × 104 cells/dish). After
an overnight attachment phase, cells were serum-starved in
MEM for
18 h before peptide addition.
Immunoprecipitations and Immunoblotting--
Cells were washed
with phosphate-buffered saline (pH 7) and then with lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM sodium orthovanadate, pH 7.4). Cells were lysed in 500 µl of lysis buffer containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, 20 µM leupeptin. After a 15-min incubation at
4 °C, the lysate was collected and centrifuged at 13,000 × g for 10 min at 4 °C to remove insoluble material. For
immunodetection of association of SHP-1 with the subunit of insulin
receptor, cells were washed in phosphate-buffered saline and
homogenized in a Dounce homogenizer with 50 mM Tris-HCl
containing 1 mM EGTA, 5 mM MgCl2,
0.5 mg/ml bacitracin, 0.03% soybean trypsin inhibitor, 5 mM sodium orthovanadate (pH 7.8). The homogenate was
centrifuged at 15,000 × g for 30 min at 4 °C. The
pellet was solubilized in 500 µl of lysis buffer for 1 h at
4 °C, and the lysate was then collected and centrifuged at
13,000 × g for 10 min at 4 °C.
Tyrosine Phosphatase Assay-- Cells were plated in 100-mm diameter dishes for 48 h, then treated with 100 nM insulin with or without 1 nM RC-160 for various times. The cells were then washed once with phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl buffer (pH 7.8) containing 2.5% CHAPS, 0.5 mg/ml cholesterol hemisuccinate, 0.05% soybean trypsin inhibitor, 140 mM NaCl, 5 mM MgCl2, 5 mM sodium orthovanadate) for 1 h at 4 °C. The lysate was clarified by centrifugation at 13,000 × g for 10 min at 4 °C. Soluble proteins (300 µg) were incubated with anti-SHP-1 antibody prebound to protein A-Sepharose for 2 h at 4 °C. The immune complexes were collected by centrifugation, washed once with a buffer (0.2% CHAPS, 0.05 mg/ml cholesterol hemisuccinate, 0.05% soybean trypsin inhibitor, 0.3% bovine serum albumin), and once with PTPase buffer (50 mM Tris, pH 7, 0.1% bovine serum albumin, 0.5 mg/ml bacitracin, 5 mM dithiothreitol). The immune complexes were then collected for PTPase activity in 300 µl of PTPase buffer. The reaction was initiated by addition of 30,000 cpm of 33P-poly(Glu, Tyr) prepared as described in Buscail et al. (15). One unit of PTPase activity was defined as the amount of the enzyme that released 1 pmol of phosphate/min at 30 °C from the radiolabeled substrate.
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RESULTS |
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Somatostatin Activates SHP-1 Activity and Dephosphorylates Insulin-mediated Tyrosine-phosphorylated SHP-1-- We previously reported that, in CHO cells expressing sst2, RC160 inhibited the mitogenic effect of insulin by a mechanism involving the stimulation of a PTPase (15). To assess whether SHP-1 plays a role in somatostatin-induced inhibition of insulin signaling, CHO/sst2 cells were incubated in the presence of 100 nM insulin with or without 1 nM RC160 for various times. SHP-1 was immunoprecipitated with anti-SHP-1 antibodies, and then the PTPase activity was examined in SHP-1 immunoprecipitates. As previously reported on the CCL39 fibroblast cell line (28), insulin caused a stimulation of SHP-1 activity in CHO/sst2 cells (Fig. 1A). A slight increase (20%, p < 0.02) was detected after 1 and 3 min of insulin treatment, with a maximal increase (70%) being observed at 10 min. Addition of RC160 in the incubation medium also resulted in an increase of SHP-1 activity, but the intensity and the time course of the stimulation were different from those observed with insulin alone. The stimulation of SHP-1 activity reached a maximum of about 170% of control as early as 1 min after RC160 addition and remained elevated for at least 10 min. The RC160-induced stimulation of SHP-1 activity was dose-dependent (Fig. 1B). It was observed at concentrations of RC160 in relation to its affinity for the sst2 receptor (15). Indeed, half-maximal and maximal stimulation occurred at 23 ± 4.9 pM and 1 nM RC160, respectively. As reported previously for the effect of analogues on tyrosine phosphatase activity (13-15), the stimulation of SHP-1 was decreased at 10 nM RC160. This could account for the coupling of sst2 to phospholipase C at concentrations higher than 1 nM (29). These results indicate that SHP-1 is a common target for insulin- as well as somatostatin-activated receptors and that activation of sst2 by RC160 leads to a rapid stimulation of SHP-1, which was more sustained and more efficient than that observed with insulin alone.
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Somatostatin Induced the Association of SHP-1 with IR--
The IR
is a heterotetrameric transmembrane tyrosine kinase. Binding of insulin
to the extracellular receptor subunit results in the rapid
autophosphorylation of tyrosine residues within the IR
, activating
receptor tyrosine kinase activity toward endogenous substrates
(31-33). These early steps of insulin action are believed to be
crucial for the propagation of the insulin signal. Since SHP-1 appears
to be a substrate of insulin receptor tyrosine kinase, we examined
whether SHP-1 interacts with IR
. The possible association of SHP-1
with IR was assessed by looking for the presence of IR
in SHP-1
immunoprecipitates from insulin-stimulated cells cultured with or
without 1 nM RC160 for various periods of time. As seen in
Fig. 3, in resting cells, there was a
detectable association of SHP-1 with IR
. Following insulin
stimulation of the cells, the amount of SHP-1 associated with IR
did
not change at 1 min of insulin treatment but increased by 2-fold above
control levels (p < 0.02) within 3-10 min of
treatment. When insulin-stimulated cells were treated with RC160, we
observed different kinetics of the association of SHP-1 with IR
.
Within 1 min of RC160 treatment, the amount of SHP-1 associated with
IR
was 3-fold higher than control (p < 0.002) and
remained elevated up to 10 min. Thus, insulin induces an association of
SHP-1 with IR
, and the major effect of RC160 is a change in the
kinetics and the magnitude of insulin-induced SHP-1-IR
interaction,
which is induced more rapidly and efficiently.
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Somatostatin Dephosphorylates Autophosphorylated IR--
Since
autophosphorylation of IR is one of the initial signaling events
after binding of insulin to its receptor and activation of its
intrinsic tyrosine kinase, we examined whether the phosphorylated IR
could be a target of SHP-1. Insulin-stimulated cells were treated or
not with 1 nM RC160, and cell lysates were
immunoprecipitated with the anti-
subunit of IR antibody and then
immunoblotted with either the polyclonal anti-IR
or the monoclonal
anti-phosphotyrosine antibody. Tyrosine phosphorylation of the 95-kDa
subunit of insulin receptor was barely detectable in control
CHO/sst2 cells but in response to insulin stimulation, IR
became
highly tyrosine phosphorylated with a maximal level at 1 min (Fig.
4). Tyrosine phosphorylation of IR
was
transient and rapidly decreased by 60-70% at 3 and 10 min
(p < 0.001). Thus the recruitment of SHP-1 to IR
was followed by the dephosphorylation of IR
. Addition of 1 nM RC160 to insulin-stimulated cells strikingly decreased the effect of insulin on IR
tyrosine phosphorylation. The time course for RC160-induced IR
dephosphorylation correlates well with
that for RC160-induced SHP-1-IR
association. Indeed, the phosphorylation of IR
was maximally reduced by 80%
(p < 0.001) at 1 min of RC160 treatment and the
inhibitory effect of RC160 was sustained up to 10 min. The effect of
RC160 was dose-dependent. Half-maximal and maximal effects
were observed with 16 ± 6 pM and 1 nM
RC160, respectively, the effect of RC160 being reversed at higher
concentration. Furthermore, RC160 inhibition of insulin-stimulated IR
tyrosine phosphorylation was reversed by pretreatment of cells with the tyrosine phosphatase inhibitor, orthovanadate, at 1 µM for 15 min (data not shown). Our results indicate
that, first, the dephosphorylation of the activated IR is correlated
with the association of SHP-1 with IR. Second, the RC160-mediated
prevention of insulin-induced tyrosine phosphorylation of IR is
time-related with the RC160-induced increase of SHP-1-IR association.
This suggests that SHP-1 is a common target of insulin and somatostatin receptors and acts as a negative regulator of IR.
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Somatostatin Dephosphorylates Insulin-induced Phosphorylated IRS-1
and Shc--
The two best characterized substrates for IR kinase
have been identified as the 185-kDa protein termed IRS-1 (insulin
receptor substrate-1) and the adaptator protein Shc (Src homology
2/
-collagen related), which are required for the recruitment of SH2
domain-containing proteins (reviewed in Refs. 34 and 35). We also next
investigated whether RC160-activated SHP-1 could affect the
phosphorylation levels of these two IR
target proteins.
Anti-phosphotyrosine immunoblots on either IRS-1 or Shc
immunoprecipitates revealed IRS-1 to be hyperphosphorylated on tyrosine
by insulin treatment (Fig. 5). The time
course of insulin-induced phosphorylation of IRS-1 was comparable to
that of IR
phosphorylation seen in Fig. 4. IRS-1 phosphorylation
peaked at 1 min and then decreased by about 50% (p < 0.001) up to 10 min. Cells treated with insulin in the presence of 1 nM RC160 exhibited reduced IRS-1 phosphorylation in
comparison with insulin alone, RC160 inducing an inhibition of about
50% of the level of insulin-stimulated IRS-1 phosphorylation, throughout the 10-min time course. When cells were preincubated with 1 µM vanadate for 15 min prior RC160 treatment, the
inhibitory effect of RC160 was completely blocked and the level of
IRS-1 tyrosine phosphorylation was similar to that observed with
insulin alone (data not shown). The decrease of IRS-1 phosphorylation in response to increasing concentrations of RC160 indicated that half-maximal and maximal effects were obtained with 14 ± 8 pM and 1 nM RC160, respectively, the effects of
RC160 being decreased at 10 nM RC160 (data not shown).
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Overexpression of Negative SHP-1(C453S) Mutant Prevents the Effect of Insulin and Somatostatin on SHP-1-- To confirm whether SHP-1 is critical for insulin- as well as somatostatin-mediated decrease of IR signaling pathway, we generated a mutated SHP-1 cDNA in which the active cysteine at position 453 was mutated to serine. This mutation results in a catalytically inactive enzyme as observed by transient expression of the SHP-1 mutant in COS-7 cells.2 We stably co-transfected the cDNA coding for the SHP-1 mutant and sst2 in CHO cells and selected the clones (CHO/sst2-SHP-1(C453S)) that expressed sst2 receptors at a similar level with that observed in CHO/sst2 cells. Furthermore, these clones overexpressed the SHP-1 mutant protein approximately 4-fold (not shown). CHO/sst2-SHP-1(C453S) cells were incubated with 100 nM insulin in the presence or absence of 1 nM RC160 for various times. SHP-1 was then immunoprecipitated with anti-SHP-1 antibodies, and the PTPase activity was measured. Insulin and RC160 no longer stimulated SHP-1 activity in cells expressing the SHP-1 mutant whatever the duration of treatment (Fig. 7).
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Overexpression of Negative SHP-1(C453S) Mutant Prevents the
Dephosphorylation of Insulin Receptor and Suppresses the Effect of
Somatostatin--
When CHO/sst2-SHP-1(C453S) cell lysates were
immunoprecipitated with the anti-IR antibody and then immunoblotted
with either the polyclonal anti-IR
or the monoclonal
anti-phosphotyrosine antibody, we observed that tyrosine
phosphorylation of the
subunit of IR was barely detectable in
control CHO/sst2-SHP-1(C453S) cells as readily observed in CHO/sst2
cells. Treatment of CHO/sst2-SHP-1(C453S) cells with insulin induced
tyrosine phosphorylation of IR
. However, this effect was not
transient and increased up to 10 min of the time course (Fig.
9). These data indicate that the
expression of the catalytically inactive SHP-1 blocks the negative
autoregulation of insulin signaling. Furthermore, expression of the
SHP-1 mutant abrogated the inhibitory effect of RC160 on
insulin-induced IR
phosphorylation, the level of IR
phosphorylation being not modified by addition of RC160 at 1 and 3 min.
At 10 min, RC160 decreased the level of IR
phosphorylation by about
50%, suggesting that another tyrosine phosphatase that negatively
regulated insulin signaling could be activated as well by RC160 in
cells expressing the SHP-1 mutant.
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Overexpression of Negative SHP-1(C453S) Mutant Abolishes the
Antiproliferative Effect of Somatostatin--
Finally, we tested the
effect of insulin and RC160 on the proliferation of
CHO/sst2-SHP-1(C453S) cells after incubation for 24 h with 1 µM insulin in the presence or not of 1 nM
RC160. As observed in Fig. 10, in cells
expressing the SHP-1 mutant and sst2, the basal proliferation was
increased by about 50% (p < 0.01) compared with
control CHO cells or CHO/sst2 cells. Furthermore, insulin stimulated
the proliferation of control cells or CHO/sst2 cells but had no more
effect in CHO/sst2-SHP-1(C453S) cells. Finally, the inhibitory effect
of RC160 was observed in CHO/sst2 cells (64%, p < 0.02) but was never seen in CHO/sst2-SHP-1(C453S) cells. These results
clearly demonstrate that SHP-1 may play a role in the negative
regulation of insulin signaling and is critical for the transduction of
the negative growth signal promoted by activation of sst2.
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DISCUSSION |
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Over the past several years much progress has been made in elucidating intracellular signaling events mediating positive growth signal by tyrosine kinase receptor activation. The processes that mediate the inactivation of these events through activation of tyrosine dephosphorylation by extracellular stimuli remain less defined. We previously demonstrated that stimulation of a membrane tyrosine phosphatase activity is an early event in the negative control of insulin-induced mitogenic signal initiated by somatostatin-activated sst2 (14). We have shown here that SHP-1 is one negative regulator of the insulin signaling pathway. Somatostatin further promotes the activation of SHP-1, leading to an earlier and more amplified dephosphorylation of IR and its downstream substrates than that compared with insulin alone.
SHP-1 has been identified as a critical negative regulator of
interleukin-3, erythropoietin, interferon-/
, and growth hormone cytokine, as well as epidermal growth factor signaling, the recruitment of the enzyme to activated membrane receptors causing dephosphorylation of the receptors or/and of downstream signaling molecules (38). Insulin
binding to its receptor results in autophosphorylation of tyrosine
residues within the
subunit of the receptor, activating intrinsic
tyrosine kinase activity toward endogenous substrates. These tyrosine
phosphorylation events are rapidly reversed, and evidence from a
variety of experimental approaches has been provided that intracellular
PTPase PTP1B and the transmembrane PTPases PTP
, PTP
, and LAR act
as negative regulators of insulin signaling by catalyzing the
dephosphorylation of insulin receptor and its substrates (39-41). The
results presented here are in favor of the role of SHP-1 in terminating
insulin signaling. Consistent with previous results on cells
overexpressing IR and SHP-1 (30), stimulation of CHO/sst2 cells by
insulin induces an increase of the SHP-1 activity which is maximal at
10 min. We further showed that SHP-1 constitutively associates with the
IR and that insulin increases the binding of the enzyme to the
autophosphorylated IR, the association reaching a maximal at 3 min and
being maintained for 10 min. The kinetics of insulin-mediated
association of SHP-1 to IR closely parallels the kinetics of decrease
of IR phosphorylation, indicating a direct correlation between
association of SHP-1 to IR, PTPase activation, and SHP-1 substrate
dephosphorylation. Such a correlation between binding and
dephosphorylation has been recently reported for the regulation of Jak2
phosphorylation by SHP-1 (42). Finally, the expression of the
catalytically inactive SHP-1 blocks the negative regulation of the
receptor autophosphorylation and induces a sustained increase of IR
tyrosine phosphorylation after insulin stimulation. Taken together,
these data indicate that IR is an early substrate of SHP-1 and that
SHP-1 may be part of the physiological negative feedback regulation of
insulin action in vivo. The precise mechanism by which SHP-1
is basely associated to IR and recruited back to activated IR requires
further definition. An association of the C-terminal region of SHP-1
with autophosphorylated IR has been reported, in vitro, with
recombinant SHP-1. However, such an association has not yet been
observed in vivo (30). A SH2-independant interaction has
been demonstrated between SHP-1 and Jak2 (42). In contrast, interaction
of SHP-1 with the erythropoietin receptor, the FC
IIB1 receptor as
well the epidermal growth factor receptor is mediated by the SH2
domains of the enzyme (24, 25, 43).
In addition, SHP-1 undergoes a rapid insulin-induced tyrosine phosphorylation in CHO-sst2 cells, in agreement with previous results (28, 30). As observed for IR and IRS-1, tyrosine phosphorylation of SHP-1 is transient, being maximal at 1 min and then decreasing by 3 min. Our data indicate that SHP-1 is an early substrate of IR and that phosphorylated SHP-1 undergoes a rapid dephosphorylation probably due to SHP-1 activation, as previously observed (44, 45). These data are strengthened by the observation that, in cells expressing the catalytically inactive SHP-1, SHP-1 is highly phosphorylated. SHP-1 has been shown to be tyrosine-phosphorylated by various growth factors and cytokines, as well as factors acting via G protein-coupled receptors (28, 30, 46-48). Two major sites for SHP-1 tyrosine phosphorylation have been identified in the C-terminal region of the molecule, Tyr536 in response to insulin in CHO cells overexpressing SHP-1 (30), Tyr564 in murine T cells, and both Tyr536 and Tyr564 in the lymphoma cell line LSTRA (45). However, the overall effect of tyrosine phosphorylation of SHP-1 is not yet elucidated. Following insulin receptor engagement, SHP-1 may become rapidly tyrosine-phosphorylated, allowing it to recruit other IR substrates and to inhibit eventually a negative insulin signaling regulator, thus contributing to IR activation.
In the other hand, we provide evidence that activation of SHP-1 following ligand occupation of somatostatin receptor sst2 is involved in the down-regulation of insulin signaling promoted by sst2. In agreement with previous results (17), the somatostatin analogue, RC160, stimulates SHP-1 activity in a time- and dose-dependent manner and at concentrations in relation with the affinity of the analogue for sst2 (15). Furthermore, we showed that somatostatin accelerates and amplifies the effect of insulin on SHP-1 activity. Indeed, somatostatin induces a more sustained and more efficient stimulation of SHP-1 than that observed with insulin alone. In addition, ligand activation of sst2 alters the rate and extent of association of SHP-1 with IR, which is induced more rapidly and efficiently than that observed with insulin alone. Consistent with the results observed upon stimulation with insulin alone, the kinetics of interaction between SHP-1 and IR induced by somatostatin closely parallel those of dephosphorylation of IR and its substrates. We previously observed that SHP-1 was constitutively associated with sst2. Ligand activation of sst2 induced an activation of SHP-1 associated with a transient increase of association of SHP-1 to sst2, which was followed by a rapid dissociation of the complex (17). Taken together, our results suggest a model for sst2 regulation of insulin signaling. Upon sst2 stimulation, activated SHP-1 dissociates from sst2, associates with IR and then dephosphorylates IR and its substrates thus leading to a negative regulation of the insulin mitogenic signaling. Consistent with this model, we observed that the expression of the catalytically inactive SHP-1 prevents somatostatin-induced activation of SHP-1 as well as somatostatin-dephosphorylation of IR and -inhibition of insulin-induced cell proliferation.
SHP-1 was found to be rapidly tyrosine-dephosphorylated by somatostatin (17). In the present study, we observed that somatostatin blocks the insulin-induced phosphorylation of SHP-1. The biological consequences of the dephosphorylation of SHP-1 on its activity are not elucidated. However, our finding argues in favor of a link between the sustained effect of somatostatin on the activation of the enzyme and its dephosphorylation. This hypothesis is strengthened by our results obtained with the catalytically inactive SHP-1, which is no longer activated by somatostatin and is stably tyrosine-phosphorylated. On the other hand, it is possible the somatostatin inhibition of insulin-induced tyrosine phosphorylation of SHP-1 may affect the interaction of the enzyme with its substrates.
Our data suggest that SHP-1 plays a role in modulating insulin-induced signaling events and are consistent with the hypothesis that somatostatin negatively regulates insulin signal transduction by controlling first the recruitment of SHP-1 to IR and its activation, causing then a dephosphorylation and an inactivation of IR and its substrates. These events lead to an inhibition of the insulin downstream signaling and the mitogenic response initiated by insulin.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. T. Reisine and Dr. M. Thomas for kindly providing CHO cells expressing sst2A receptors and SHP-1 cDNA, respectively and to Dr. C. Nahmias for fruitful discussions.
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FOOTNOTES |
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* This work was supported by grants from Association Pour la Recherche Contre le Cancer (6755), Conseil Régional Midi-Pyrénées (9407556), and Ligue Nationale Contre le Cancer (257 3D129B).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.
The first two authors contributed equally to this work.
¶ To whom correspondence should be addressed: INSERM U151, Institut Louis Bugnard, IFR 31, CHU Rangueil, 31403 Toulouse Cedex 04, France. Tel.: 33 5 61 32 24 07; Fax: 33 5 61 32 24 03; E-mail: susinich{at}rangueil.inserm.fr.
1
The abbreviations used are: PTPase, protein
tyrosine phosphatase; CHO, Chinese hamster ovary; IR, insulin receptor;
IR, insulin receptor subunit
; IRS-1, insulin receptor substrate
1;
MEM,
-minimal essential medium; PAGE, polyacrylamide gel
electrophoresis; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2 C. Nahmias, unpublished results.
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REFERENCES |
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