sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1.

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.

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)(23)(24)(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.

MATERIALS AND METHODS
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 g/ml. Geneticinresistant clones expressing sst2 (CHO/sst2) were screened for somatostatin binding using [ 125 I-Tyr 11 ]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 5Ј-GAT GCC AGC GCT GGA ATG CAC AAT-3Ј 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 g/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.
For cell growth assay, cells were cultured in ␣MEM supplemented with 10% fetal calf serum and plated in 35-mm diameter dishes at 75 ϫ 10 3 cells/ml (2 ml/dish). After an attachment phase, cells were cultured in serum-free medium overnight (time 0), and then medium was replaced by fresh ␣MEM containing 100 nM insulin with or without different concentrations of RC-160. Cell growth was measured after a 24-h treatment by cell counting with a Coulter counter model ZM (Coulter Electronics) as described previously (15).
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 Na 4 P 2 O 7 , 100 mM NaF, 2 mM sodium orthovanadate, pH 7.4). Cells were lysed in 500 l of lysis buffer containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 20 M leupeptin. After a 15-min incubation at 4°C, the lysate was collected and centrifuged at 13,000 ϫ 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 MgCl 2 , 0.5 mg/ml bacitracin, 0.03% soybean trypsin inhibitor, 5 mM sodium orthovanadate (pH 7.8). The homogenate was centrifuged at 15,000 ϫ g for 30 min at 4°C. The pellet was solubilized in 500 l of lysis buffer for 1 h at 4°C, and the lysate was then collected and centrifuged at 13,000 ϫ g for 10 min at 4°C.
Proteins of interest were immunoprecipitated with either monoclonal antibodies coupled to protein G-Sepharose or polyclonal antibodies coupled to protein A-Sepharose during 2 h at 4°C with gentle rocking. The immune complexes were collected by centrifugation, washed twice with a 30 mM HEPES buffer containing 30 mM NaCl, 0.1% Triton X-100, pH 7.4), boiled for 5 min in 50 l of sample buffer (4.5 mM Na 2 HPO 4 , 2.7%, SDS, 9% glycerol, 10% bromphenol blue/␤-mercaptoethanol). The proteins were then separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride or nitrocellulose membranes. Membranes were incubated overnight at 4°C in saline buffer (1 mM Tris-HCl, 14 mM NaCl, pH 7.4) containing 5% non-fat dry milk and blotted with specific antibodies for 3 h at room temperature. Immunoreactive bands were detected by ECL immunodetection system (Amersham Corp.).
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 MgCl 2 , 5 mM sodium orthovanadate) for 1 h at 4°C. The lysate was clarified by centrifugation at 13,000 ϫ g for 10 min at 4°C. Soluble proteins (300 g) 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 l of PTPase buffer. The reaction was initiated by addition of 30,000 cpm of 33 P-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.

RESULTS
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 Ͻ 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 Ϯ 4.9 pM and 1 nM RC160, respectively. As reported previously for the effect of analogues on tyrosine phosphatase activity (13)(14)(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.
Several growth factors are known to induce the tyrosine phosphorylation of SHP-1, and insulin has been shown to phosphorylate SHP-1 on Tyr 536 in a lymphoma cell line (30). To determine the effect of insulin and somatostatin on the level of tyrosine phosphorylation of SHP-1, we performed Western blotting of SHP-1 immunoprecipitates with either anti-phosphotyrosine or anti-SHP-1 antibodies. Our immunoblots revealed that SHP-1 was rapidly tyrosine-phosphorylated in response to insulin (Fig. 2). Maximal stimulation of SHP-1 tyrosine phosphorylation by insulin (2-fold increase) was reached at 1 min and decreased up to 10 min in our time course. Addition of RC160 to insulin-stimulated cells completely suppressed the stimulatory effect of insulin on SHP-1 tyrosine phosphorylation. A maximal inhibitory effect of RC160 was observed at 3 min, the level of tyrosine phosphorylation of SHP-1 being significantly lower than the control level (p Ͻ 0.002). These results indicate that SHP-1 is tyrosine-phosphorylated following the activation of the insulin receptor and that this effect is suppressed by RC160.
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 Ͻ 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 Ͻ 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.
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 Ͻ 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 Ͻ 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 Ϯ 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 M 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.
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 RC160activated 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 Ͻ 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 10min time course. When cells were preincubated with 1 M 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 Ϯ 8 pM and 1 nM RC160, respectively, the effects of RC160 being decreased at 10 nM RC160 (data not shown).
We then examined whether the phosphorylation of Shc was also altered by insulin and RC160. Shc is an SH2 domaincontaining protein found as two dominant, widely expressed forms of 46 and 52 kDa as well as a 66-kDa form with a more restricted expression. Shc proteins act as adaptator proteins in signaling pathways generating from diverse stimuli. Shc phosphorylation was detected in the basal state, and insulin stimulated by about 2-fold the tyrosine phosphorylation of Shc with a slower time course compared with that of IR␤ and IRS-1 as previously reported (36,37). Maximal phosphorylation occurred at 3 min and was decreased at 10 min (p Ͻ 0.001) (Fig.  6). Addition of RC160 to the culture medium resulted in a decrease of insulin-stimulated Shc tyrosine phosphorylation

FIG. 4. Effect of insulin and RC160 on tyrosine phosphorylation of IR in CHO/sst2 cells.
A, serum-starved CHO/sst2 cells were incubated at 37°C for the indicated times with 100 nM insulin with (IϩR) or without (I) 1 nM RC160 or were not treated (Cont, control) prior to solubilization. Cell lysates were subjected to immunoprecipitation with anti-IR␤ (Ip: IR␤) antibodies. Immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine antibodies (blot: P-Tyr). The same filter was then reprobed with anti-IR␤ antibodies (blot: IR␤). Arrows indicate the position of IR␤. B, immunoblots were densitometrically analyzed, and data were plotted as percentage of maximal values obtained from cells treated for 1 min with insulin, which were taken as 100%. Data from three separate experiments are presented as means Ϯ S.E. (Statistical comparison by Student's t test, insulin ϩ RC160 versus insulin, p Ͻ 0.001 at 1 min, p Ͻ 0.05 at 3 and 10 min.) sst2 Activation of SHP-1 and Inhibition of Insulin Signaling which reached 50% (p Ͻ 0.001) at 3 min. RC160-induced inhibition of Shc phosphorylation was suppressed by preincubation of cells for 15 min with 1 M orthovanadate before RC160 treatment. The RC160-induced dephosphorylation of Shc was dose-dependent with half-maximal and maximal effects observed with 23 Ϯ 2.5 pM and 1 nM, respectively. At higher concentration of RC160, the effect of RC160 was reversed (data not shown). All these results showed that RC160 negatively regulates insulin signaling by dephosphorylating the IR␤ and some of its key downstream targets. Furthermore, these last events may involve the activation of a tyrosine phosphatase.

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 somatostatinmediated 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).
In addition, SHP-1 protein was 2-fold more phosphorylated on tyrosine residues in CHO/sst2-SHP-1(C453S) cells compared with CHO/sst2 cells in basal conditions (not shown). However, the phosphatase was not further phosphorylated upon insulin stimulation. Remarkably, addition of RC160 did not cause any dephosphorylation of SHP-1, indicating that the blockade of SHP-1 catalytic activity renders the enzyme refractory to its regulation by insulin or RC160 (Fig. 8).

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.
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/ 2 C. Nahmias, unpublished results. sst2 Activation of SHP-1 and Inhibition of Insulin Signaling lation of insulin signaling and is critical for the transduction of the negative growth signal promoted by activation of sst2. DISCUSSION 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 insulinmediated 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, Tyr 536 in response to insulin in CHO cells overexpressing SHP-1 (30), Tyr 564 in murine T cells, and both Tyr 536 and Tyr 564 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 sst2 Activation of SHP-1 and Inhibition of Insulin Signaling 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.