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Endocrinology Vol. 140, No. 10 4585-4594
Copyright © 1999 by The Endocrine Society

ARTICLES

Role of the Src Homology 2 (SH2) Domain and C-Terminus Tyrosine Phosphorylation Sites of SH2-Containing Inositol Phosphatase (SHIP) in the Regulation of Insulin-Induced Mitogenesis1

Tsutomu Wada, Toshiyasu Sasaoka, Manabu Ishiki, Hiroyuki Hori, Tetsuro Haruta, Hajime Ishihara and Masashi Kobayashi

First Department of Medicine, Toyama Medical and Pharmaceutical University, Toyama, 930-0194 Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., First Department of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194, Japan. E-mail: tsasaoka-tym{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the role of SHIP in insulin-induced mitogenic signaling, we used a truncated SHIP lacking the SH2 domain ({Delta}SH2-SHIP) and a Y917/1020F-SHIP (2F-SHIP) in which two tyrosines contributing to Shc binding were mutated to phenylalanine. Wild-type (WT)-, {Delta}SH2-, and 2F-SHIP were transiently transfected into Rat1 fibroblasts overexpressing insulin receptors (HIRc). Insulin-stimulated tyrosine phosphorylation of WT-SHIP and {Delta}SH2-SHIP, whereas tyrosine phosphorylation of 2F-SHIP was not detectable, indicating that 917/1020-Tyr are key phosphorylation sites on SHIP. Although SHIP can bind via its 917/1020-Tyr residues and SH2 domain to Shc PTB domain and 317-Tyr residue, respectively, insulin-induced SHIP association with Shc was more greatly decreased in 2F-SHIP cells than that in {Delta}SH2-SHIP cells. Insulin stimulation of Shc association with Grb2, which is important for p21ras-MAP kinase activation, was decreased by overexpression of WT- and 2F-SHIP. Importantly, insulin-induced Shc·Grb2 association was not detectably reduced in {Delta}SH2-SHIP cells. In accordance with the extent of Shc association with Grb2, insulin-induced MAP kinase activation was relatively decreased in both WT-SHIP and 2F-SHIP cells, but not in {Delta}SH2-SHIP cells. To examine the functional role of SHIP in insulin’s biological action, insulin-induced mitogenesis was compared among these transfected cells. Insulin stimulation of thymidine incorporation and bromodeoxyuridine incorporation was decreased in WT-SHIP cells compared with that of control HIRc cells. Expression of 2F-SHIP also significantly reduced insulin-induced mitogenesis, whereas it was only slightly affected by overexpression of {Delta}SH2-SHIP. Furthermore, the reduction of insulin-induced mitogenesis in WT-SHIP cells was partly compensated by coexpression of Shc. These results indicate that SHIP plays a negative regulatory role in insulin-induced mitogenesis and that the SH2 domain of SHIP is important for its negative regulatory function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADAPTOR PROTEIN Shc is a key molecule that plays a role in insulin-induced mitogenesis, largely through the activation of p21ras protein (1, 2, 3, 4, 5). Shc is composed of an amino-terminal phosphotyrosine binding (PTB) domain, a collagen homology (CH) domain, and a carboxyl-terminal src homology 2 (SH2) domain (1). Following insulin stimulation, Shc is tyrosine phosphorylated mainly on 317-Tyr residue within the CH domain, and subsequently associates via the tyrosine phosphorylated residue with the SH2 domain of Grb2 (6, 7, 8). Grb2 exists as a preformed complex with Sos, which is a guanine nucleotide exchange factor for p21ras (5, 9, 10, 11). Shc binding to the Grb2·Sos complex is thought to be important for the membrane localization of Sos, where it leads to p21ras activation (5, 9, 10, 11). In addition to the interaction with Grb2, Shc has been shown to associate with src homology 2 containing 5'-phosphatase (SHIP) (12, 13, 14, 15, 16, 17). SHIP contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and two phosphotyrosine binding (PTB) consensus sequences and a proline rich region at the carboxyl tail (12, 13, 14). SHIP selectively hydrolyzes the 5'-phosphate from Ins (1, 3, 4, 5) P4 and PtdIns (3, 4, 5) P3, which are implicated in growth factor-mediated signaling (12, 13, 14, 18). Although the precise mechanisms regulating inositol lipids to affect downstream signaling remains unclear, SHIP has been implicated in the negative regulation of Fc{gamma}RIIB receptor-mediated B cell proliferation and signaling in mast cells (19, 20, 21, 22). Furthermore, important role of SHIP-mediated dephosphorylation of PtdIns (3, 4, 5) P3 in down-regulating insulin signaling was suggested by the fact that insulin-induced Xenopus oocyte maturation was inhibited by SHIP expression and that the inhibitory function of SHIP was dependent on its phosphatase activity (23).

In addition to the role of SHIP dependent on its catalytic activity, it is possible that, via its SH2 domain or carboxyl-terminus tyrosine residues, SHIP could also regulate other signaling molecules. For example, SHIP may influence p21ras activation through inhibiting Shc·Grb2 pathway becaues SHIP can associate with Shc. In this regard, SHIP SH2 domain and carboxyl-terminal tyrosine residues can interact with Shc 317-Tyr residue and the PTB domain, respectively (16, 17). SHIP SH2 domain interaction with Shc is assumed to affect insulin-mediated Shc·Grb2 pathway by competing with Shc for binding to Grb2 (16). In addition, SHIP may affect Shc·Grb2 pathway by modulating insulin-induced tyrosine phosphorylation of Shc, because SHIP is also capable of binding via its carboxyl-terminus tyrosine residues to Shc PTB domain, which interact with the activated insulin receptor (7, 17, 24). To clarify the role of SHIP SH2 domain and carboxyl-terminus phosphotyrosine binding consensus sites in the regulation of insulin-induced mitogenesis, we used a truncated SHIP lacking the SH2 domain ({Delta}SH2-SHIP) and a Y917/1020F-SHIP (2F-SHIP) in which carboxyl-terminus two tyrosine residues were replaced with phenylalanine. Wild-type (WT)-SHIP, {Delta}SH2-SHIP, and 2F-SHIP were transiently transfected into Rat1 fibroblasts overexpressing insulin receptors (HIRc). Insulin-induced Shc tyrosine phosphorylation and Shc·Grb2 association for p21ras-MAP kinase activation leading to cell cycle progression was compared among the transfected cells. These experiments reveal a key role for the SH2 domain of SHIP in negatively influencing the insulin-induced mitogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Porcine insulin was the kind gift of Shimizu Pharmaceutical Co. (Shizuoka, Japan). Effectene transfection reagents were purchased from QIAGEN Inc. (Valencia, CA). [3H]Thymidine was from NEN Life Science Products. A polyclonal anti-Shc antibody and a monoclonal anti-Grb2 antibody were from Transduction Laboratories (Lexington, KY). A polyclonal anti-GST antibody, a polyclonal anti-SHIP antibody, a monoclonal anti-Shc antibody, and a monoclonal antiphosphotyrosine antibody (PY99) were from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal antiphospho-specific p44/p42 MAP kinase (Thr202/Tyr204) antibody and a polyclonal anti-MAP kinase antibody were from New England Biolabs, Inc. (Beverly, MA). Bromodeoxyuridine (BrdU), a monoclonal anti-BrdU antibody, and enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech Corp. (Uppsala, Sweden). All other routine reagents were analytical grade and purchased from Sigma (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Cell culture
Rat1 fibroblasts overexpressing 1 x 106 human insulin receptors per cell (HIRc) were kindly supplied from Dr. J. M. Olefsky (University of California San Diego, San Diego, CA). HIRc cells were maintained in DMEM/F12 medium supplemented with 10% FCS as previously described (25).

Plasmid
DNA encoding GST-tagged wild-type Shc and SHIP were generated by PCR and subcloning of DNA fragments into the pEBG vector as described previously (17, 26, 27). GST tagged {Delta}SH2-SHIP, which lacks the SH2 domain of SHIP ({Delta}SH2-SHIP) and 2F-SHIP, in which tyrosines 917 and 1020 of murine SHIP have been replaced by phenylalanine (2F-SHIP) were generated by PCR-based mutagenesis as described previously (17, 26, 28). These mutant SHIP DNA were also subcloned into the pEBG vector. All constructs were sequenced, and the presence of appropriate mutations was confirmed.

DNA transfection
Transient transfection into HIRc cells was performed using the Effectene transfection reagents according to the manufacturer’s instructions (QIAGEN Inc.). In brief, the cells were washed twice with sterile PBS followed by addition of 1.6 ml DMEM to each 35-mm well. Preformed complexes (Effectene reagents including 0.8 µg of the indicated DNA per well) were then added to each well and dishes were placed at 37 C in 5% CO2. Approximately 48 h post transfection, the cells expressing equivalent levels of various SHIP proteins were used for further studies. The transfection efficiency of the SHIP constructs was about 45% by the immunofluorescent staining with anti-GST antibody as described below, and the efficiency was not significantly different among the expression of various SHIP constructs.

Immunoprecipitations and Western blotting
Cells were serum-starved for 24 h in DMEM. The cells were treated with 17 nM insulin at 37 C for the indicated times. The cells were lysed in a buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 160 mM sodium fluoride, 10 µM aprotinin, 10 µM leupeptin, pH 7.4, for 15 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. The supernatants (100 µg of protein) were immunoprecipitated with the indicated antibodies or precipitated with glutathione Sepharose beads for 2 h at 4 C. The precipitates were then separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a Bio-Rad Laboratories, Inc. Transblot apparatus. The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA, pH7.5, for 2 h at 20 C. The membranes were then probed with the specified antibodies for 2 h at 20 C. After washing the membranes in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5, blots were incubated with horseradish peroxidase-linked second antibody followed by enhanced chemiluminescence detection using ECL reagent according to the manufacturer’s instructions (Amersham Pharmacia Biotech). To ensure equal amount of protein loaded for the study, the cell lysates were immunoblotted with anti-Shc antibody or anti-MAP kinase antibody. In addition, comparable expression of SHIP constructs among the transfected cells was also confirmed by immunoblotting of the cell lysates with anti-GST antibody.

Thymidine incorporation
Cells were serum-starved for 20 h. After stimulation of the cells with various concentrations of insulin for 20 h, 1 µCi of [3H]thymidine was added for further 4 h. The cells were washed twice with ice-cold PBS, twice with ice-cold 10% trichloroacetic acid, and once with 95% ethanol. Then the cells were dissolved in 0.2 N NaOH and 0.2% SDS, and counted in a liquid scintillation counter (29). In these thymidine incorporation studies, comparable expression of SHIP constructs was ensured by immunoblotting of the cell lysates obtained from separate set of the transfected cells with anti-GST antibody.

BrdU incorporation and immunostaining
Cells grown on glass coverslips were transfected with various SHIP constructs. The transfected cells were rendered quiescent by starvation for 24 h. Serum-starved cells were incubated with BrdU plus 1.7 nM insulin for 16 h at 37 C. The cells were fixed with acid alcohol (90% ethanol, 5% acetic acid) for 20 min at 22 C and then incubated with mouse monoclonal anti-BrdU antibody for 1 h at 22 C. The cells were then stained by incubation with rhodamine-labeled donkey antimouse IgG antibody and fluorescein isothiocyanate-labeled donkey anti-GST antibody for 1 h at 22 C. After the coverslips were mounted, the cells were analyzed with a Microphot-FXA fluorescence microscope (Nikon, Tokyo, Japan) (4).

Statistical analysis
The data are represented as means ± SE. P values were determined by unpaired Student’s t test, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of GST-tagged SHIP constructs
To examine the role of SHIP in insulin signaling, we transiently transfected pEBG (empty vector), wild-type SHIP (WT-SHIP), a truncated SHIP lacking SH2 domain ({Delta}SH2-SHIP), and a Y917/1020F-SHIP (2F-SHIP) in which key tyrosine phosphorylation sites of SHIP were mutated to phenylalanine, into Rat1 fibroblasts overexpressing human insulin receptors (HIRc) (Fig. 1AGo). After expression of comparable amounts of these SHIP constructs was confirmed by immunoblotting of the cell lysates with anti-SHIP antibody, we used this transient expression procedure for further studies (Fig. 1BGo). Because the expression of endogenous SHIP is low, only a faint band of endogenous SHIP was seen by the immunoblotting of the membranes with anti-SHIP antibody (data not shown).



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  		Figure 1. Construction of expression plasmids and expression of wild-type and mutant SHIP. A, WT-SHIP, {Delta}SH2-SHIP, and 2F-SHIP cDNA were subcloned into pEBG vector. Schematic structures of SHIP are shown and all constructs have GST-tag at the N terminus of SHIP. B, Cells transfected with either pEBG alone, WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP were immunoprecipitated with anti-SHIP antibody. The precipitates were subjected to SDS-PAGE and immunoblotted with anti-GST antibody.

 
Insulin-induced tyrosine phosphorylation of SHIP in the transfected cells
It has been reported that SHIP is tyrosine phosphorylated following cytokine stimulations in hematopoietic cells (30, 31, 32). Likewise, cross-linking of the B-cell antigen receptor or T-cell activation also induces tyrosine phosphorylation of SHIP (33, 34). Therefore, we examined the effect of insulin on SHIP tyrosine phosphorylation in the transfected cells. As shown in Fig. 2AGo, insulin-stimulated tyrosine phosphorylation of WT-SHIP was peaked at 1 min after insulin treatment, and declined thereafter. {Delta}SH2-SHIP expression also gave similar amounts of insulin-induced tyrosine phosphorylation, although the time course was somewhat delayed compared with WT-SHIP expression (Fig. 2BGo). In contrast, insulin stimulation did not elicit detectable tyrosine phosphorylation of 2F-SHIP (Fig. 2AGo). These data suggest that two tyrosines (Y917/Y1020) at SHIP carboxyl-terminus represent critical phosphorylation sites and that SHIP SH2 domain may also play a role in the kinetics of insulin-induced SHIP tyrosine phosphorylation.



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  		Figure 2. Time course of insulin-stimulated tyrosine phosphorylation of SHIP in the transfected cells. A, HIRc cells were transfected with either pEBG alone, WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP. The cells were serum-starved and then treated with 17 nM insulin for the indicated times. The cells were solubilized, and the cell lysates were precipitated with glutathione Sepharose beads. The precipitates were subjected to SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody. B, The amount of tyrosine phosphorylated SHIP was quantitated by densitometry and presented as the percentage of SHIP tyrosine phosphorylation seen at 1 min after insulin stimulation in the WT-SHIP transfected cells. The results are the mean ± SE of four separate experiments.

 
Insulin-induced SHIP association with Shc in the transfected cells
SHIP has been shown to be capable of interacting with Shc 317-Tyr and the PTB domain via SHIP SH2 domain and the carboxyl-terminus two tyrosines, respectively, after cytokine stimulations (16, 17, 35). We therefore examined the role of SHIP SH2 domain and the carboxyl-terminus two tyrosines in the interaction with Shc following insulin stimulation as shown in Fig. 3AGo. SHIP was minimally associated with Shc in the basal state of any transfected cells. Following insulin stimulation, WT-SHIP association with Shc was increased by 5 min, and gradually decreased thereafter. Insulin-induced SHIP association with Shc was also observed in {Delta}SH2-SHIP and 2F-SHIP cells, although the amount was decreased compared with that in WT-SHIP cells. Importantly, the amount of the association was more greatly reduced in 2F-SHIP cells compared with that in {Delta}SH2-SHIP cells. After 5 min of insulin stimulation, SHIP association with Shc was decreased to 58 ± 5% and 78 ± 4% in 2F-SHIP and {Delta}SH2-SHIP cells, respectively, compared with that in WT-SHIP cells as shown in Fig. 3BGo. These results suggest that although SHIP has two interacting sites for Shc via either SHIP-SH2 domain or SHIP carboxyl-terminus tyrosine residues, the latter being more predominant following insulin stimulation.



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  		Figure 3. Insulin-induced SHIP association with Shc in the transfected cells. A, HIRc cells were transfected with either pEBG alone, WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP. The cells were serum-starved and then treated with 17 nM insulin for the indicated times. The cells were solubilized, and the cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates were subjected to SDS-PAGE, and immunoblotted with anti-GST antibody. B, The amount of SHIP associated with Shc was quantitated by densitometry and presented as the percentage of the amount of SHIP associated with Shc seen at 5 min after insulin stimulation in the WT-SHIP transfected cells. The results are the mean ± SE of four separate experiments.

 
Tyrosine phosphorylation of Shc was not affected by expression of SHIP
Shc binds, via its PTB domain, to the activated insulin receptor ß-subunit and also to SHIP carboxyl-terminus two phosphotyrosine binding consensus sites (7, 24). Therefore, expression of SHIP might affect insulin-induced tyrosine phosphorylation of Shc. To address this issue, insulin-stimulated tyrosine phosphorylation of Shc was compared among the SHIP transfected cells. As shown in Fig. 4AGo, insulin-induced tyrosine phosphorylation of Shc in a time-dependent manner in original HIRc cells. Importantly, the kinetics of insulin-induced Shc tyrosine phosphorylation was not affected by expression of either WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP. These results with the time-course of Shc tyrosine phosphorylation are summarized in Fig. 4BGo. Following insulin stimulation, tyrosine phosphorylation of Shc was comparable among Mock, WT-SHIP, {Delta}SH2-SHIP, and 2F-SHIP transfected cells. These results indicate that SHIP expression does not inhibit Shc tyrosine phosphorylation.



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  		Figure 4. Effects of SHIP expression on insulin-induced tyrosine phosphorylation of Shc. A, HIRc cells were transfected with either pEBG alone, WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP. The cells were serum-starved and then treated with 17 nM insulin for the indicated times. The cells were solubilized, and the cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates were subjected to SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody. B, The amount of tyrosine phosphorylation of Shc was quantitated by densitometry and presented as the percentage of Shc tyrosine phosphorylation seen 15 min after insulin stimulation in the Mock transfected cells. The results are the mean ± SE of four separate experiments.

 
Shc association with Grb2 was decreased by expression of SHIP
Tyrosine phosphorylated Shc binds to Grb2, and overexpression of Shc has been shown to increase insulin-induced Shc association with Grb2 (8). Because phosphorylated 317-Tyr residue of Shc can bind to the SH2 domain of both Grb2 and SHIP, SHIP expression may compete with Shc for Grb2 binding. Therefore, insulin-induced Shc association with Grb2 was next examined in the SHIP-transfected cells. As shown in Fig. 5AGo, after insulin stimulation, Shc associated with Grb2 in a time-dependent manner in the mock transfected HIRc cells. Expression of WT-SHIP led to a decrease in the Shc·Grb2 association. Although insulin-induced Shc association with Grb2 was also decreased by expression of 2F-SHIP, it was not significantly affected by expression of {Delta}SH2-SHIP. These results with the time-course of Shc association with Grb2 are summarized in Fig. 5BGo. Following 5 min of insulin stimulation, the Shc·Grb2 association was decreased to 68 ± 2% and 71 ± 3% by expression of WT-SHIP and 2F-SHIP, respectively, compared with that in HIRc cells. In contrast, the Shc·Grb2 association was not apparently affected in {Delta}SH2-SHIP cells. To ensure equal Shc immunoprecipitation, the anti-Shc immunoprecipitates were also immunoblotted with anti-Shc antibody (Fig. 5CGo). In addition, comparable expression of SHIP constructs among the transfected cells was confirmed by immunoblotting of the cell lysates with anti-GST antibody (Fig. 5DGo). These data suggest that SHIP competes via its SH2 domain with Shc for binding to Grb2.



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  		Figure 5. Effects of SHIP expression on insulin-induced Shc association with Grb2. A, HIRc cells were transfected with either pEBG alone, WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP. The cells were serum-starved and then treated with 17 nM insulin for the indicated times. The cells were solubilized, and the cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates were subjected to SDS-PAGE, and immunoblotted with anti-Grb2 antibody. B, The amount of Grb2 associated with Shc was quantitated by densitometry and presented as the percentage of Shc·Grb2 association seen at 5 min after insulin stimulation in the Mock transfected cells. The results are the mean ± SE of five separate experiments. C, The cell lysates were immunoprecipitated with anti-Shc antibody and immunoblotted with anti-Shc antibody. D, The cell lysates were immunoblotted with anti-GST antibody.

 
Effect of SHIP expression on insulin-induced MAP kinase activation
Shc·Grb2 binding has been shown to be important for MAP kinase activation (3, 4, 5, 7, 8, 9, 10, 11). Because phosphorylation of both tyrosine and threonine residues of MAP kinase is implicated in the activation (36, 37), we examined the effect of SHIP expression on insulin-induced MAP kinase activation using phospho-specific MAP kinase antibody. As can be seen in Fig. 6AGo, insulin activated MAP kinase in a time-dependent manner. In accordance with the results of Shc·Grb2 association, expression of WT-SHIP led to a decreased insulin-activated MAP kinase. Likewise, the MAP kinase activity was significantly reduced by expression of 2F-SHIP. In contrast, insulin-induced MAP kinase activation was not apparently impaired by expression of {Delta}SH2-SHIP. The time course of insulin-stimulated MAP kinase activation is summarized in Fig. 6BGo. After 5 min of insulin stimulation, expression of WT-SHIP and 2F-SHIP resulted in a modest reduction of MAP kinase activity to 68 ± 3% and 70 ± 2%, respectively, compared with the mock transfected HIRc cells. In contrast, expression of {Delta}SH2-SHIP decreased it only to 89 ± 3%. To ensure equal amount of protein used for the study, the cell lysates were immunoblotted with anti-MAP kinase antibody (Fig. 6CGo). In addition, comparable expression of SHIP constructs among the transfected cells was also confirmed by immunoblotting of the cell lysates with anti-GST antibody (Fig. 6DGo).



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  		Figure 6. Insulin-induced MAP kinase activity in the SHIP transfected cells. A, HIRc cells were transfected with either pEBG alone, WT-SHIP, {Delta}SH2-SHIP, or 2F-SHIP. The cells were serum-starved and then treated with 17 nM insulin for the indicated times. The cells were solubilized, and the cell lysates were subjected to SDS-PAGE. The sample was then immunoblotted with antiphosphospecific p44/p42 MAP kinase antibody. B, The amount of phosphorylated MAP Kinase was quantitated by densitometry and presented as the percentage of phosphorylated MAP kinase seen at 5 min after insulin stimulation in the Mock transfected cells. The results are the mean ± SE of four separate experiments. C, The cell lysates were immunoblotted with anti-MAP kinase antibody. D, The cell lysates were immunoblotted with anti-GST antibody.

 
Insulin-induced thymidine incorporation in the transfected cells
We have reported that overexpression of Shc increases insulin-induced Shc association with Grb2 resulting in enhanced MAP kinase activation and thymidine incorporation (8). Because SHIP appears to compete, via its SH2 domain, with Shc for binding to Grb2 leading to MAP kinase activation, thymidine incorporation was assayed in the SHIP-transfected cells as shown in Fig. 7Go. Insulin stimulated thymidine incorporation in a dose-dependent manner with an ED50 value of 1.3 ± 0.2 nM in HIRc cells transfected with pEBG vector alone. Expression of WT-SHIP resulted in decreased insulin sensitivity with a rightward shift of the dose-response curve (ED50 value, 6.0 ± 0.5 nM; P < 0.001 vs. Mock). Insulin sensitivity was also significantly reduced by expression of 2F-SHIP (ED50 value, 5.2 ± 0.4 nM; P < 0.005 vs. Mock). In contrast, insulin-stimulated thymidine incorporation was only slightly affected and the reduction of insulin sensitivity was not apparent by expression of {Delta}SH2-SHIP (ED50 value, 1.9 ± 0.4 nM; p = N.S.). The maximum values of insulin-stimulated thymidine incorporation were also relatively decreased in WT-SHIP and 2F-SHIP cells compared with that in Mock and {Delta}SH2-SHIP cells. However, the difference in insulin responsiveness was not statistically significant among the transfected cells.



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  		Figure 7. Insulin-induced thymidine incorporation in the SHIP transfected cells. HIRc cells were transfected with either pEBG alone(•), WT-SHIP(), {Delta}SH2-SHIP({square}), or 2F-SHIP({triangleup}). Insulin stimulation of thymidine incorporation in the transfected cells was measured as described in Experimental procedures. Dose-response curves of insulin-stimulated thymidine incorporation are shown. Results are expressed as the percent maximum stimulation and are the mean ± SE of four separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: pEBG, b = 1614 ± 131 dpm/1 x 106 cells and m = 5826 ± 341; WT-SHIP, b = 1663 ± 169 and m = 4605 ± 400; {Delta}SH2-SHIP, b = 1646 ± 151 and m = 5314 ± 194; 2F-SHIP, b = 1585 ± 125 and m = 4786 ± 318.

 
Effect of coexpression of Shc and SHIP on insulin-induced thymidine incorporation
Because expression of SHIP decreased insulin sensitivity for insulin-induced thymidine incorporation because of possible sequestration of Shc from binding to Grb2, coexpression of Shc might compensate the reduced insulin sensitivity by SHIP expression. To address this issue, insulin-induced thymidine incorporation was examined by coexpression of WT-Shc and WT-SHIP. Transient expression of Shc led to enhanced insulin sensitivity with an ED50 value of 0.8 ± 0.1 nM (P < 0.05 vs. Mock) as we previously reported in stably Shc overexpressed cells (8). Although expression of WT-SHIP decreased insulin sensitivity for insulin-induced thymidine incorporation, cotransfection of Shc with WT-SHIP partly compensate the decreased insulin sensitivity by expression of WT-SHIP alone, and the insulin sensitivity was partly improved with an ED50 value of 2.5 ± 0.5 nM (Fig. 8Go). These data further indicate that SHIP is a negatively regulating molecule by competing with Shc to mediate insulin’s mitogenic effects.



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  		Figure 8. Effect of cotransfection of Shc and SHIP on insulin-induced thymidine incorporation. HIRc cells were transfected with pEBG(•), WT-SHIP(), Shc({blacksquare}), or WT-SHIP and Shc({blacktriangleup}). Insulin stimulation of thymidine incorporation in the transfected cells was measured as described in Experimental procedures. Dose-response curves of insulin-stimulated thymidine incorporation are shown. Results are expressed as the percent maximum stimulation and are the mean ± SE of four separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: pEBG, b = 1614 ± 131 dpm/1 x 106 cells and m = 5826 ± 341; WT-SHIP, b = 1663 ± 169 and m = 4605 ± 400; WT-Shc, b = 2256 ± 86 and m = 6215 ± 321; WT-SHIP and WT-Shc, b = 2172 ± 168 and m = 5297 ± 309.

 
Insulin-induced BrdU incorporation in the transfected cells
Because the transfection efficiency in the study may affect the results with thymidine incorporation studies, we also assessed insulin-induced mitogenesis by the independent approach of BrdU incorporation as shown in Fig. 9Go. BrdU incorporation in the cells that are transfected with various SHIP constructs can be unambiguously detectable by examing the BrdU labeling combined with cytoplasmic immunofluorescent staining of the tagged exogenous SHIP proteins. The study with BrdU incorporation showed the basically same results as the results with thymidine incorporation studies. Basal BrdU incorporation was 10~15%, and it was not significantly different among the transfected cells. Insulin-induced BrdU incorporation to 64 ± 3% in the Mock transfected cells. Expression of Shc increased insulin-stimulated BrdU incorporation to 74 ± 1%. In agreement with the results of thymidine incorporation studies, insulin-stimulated BrdU incorporation was decreased to 35 ± 6% and 41 ± 6% by expression of WT-SHIP and 2F-SHIP, respectively. In addition, expression of {Delta}SH2-SHIP only slightly decreased insulin-stimulated BrdU incorporation to 60 ± 3%. Furthermore, the reduction of BrdU incorporation by expression of WT-SHIP was again compensated by coexpression of Shc, and 63 ± 1% of the cells were positive for BrdU incorporation by cotransfection of WT-SHIP and Shc.



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  		Figure 9. BrdU incorporation in the transfected cells. BrdU incorporation in the transfected cells was assayed as described in Experimental procedures. Results are expressed as the percent of total transfected cells and are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-stimulated BrdU incorporation in Mock transfected cells by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of phosphatidylinositol (PI) 3'-kinase by receptors for growth factors including insulin has been implicated as a critical step in mitogenic signaling (38, 39, 40). Previous studies showed that blockade of PI3'-kinase function either by expression of dominant negative p85 regulatory subunit of PI3'-kinase, by microinjection of N-terminal SH2 domain of p85, or by employment with pharmacological inhibitors inhibited insulin-induced mitogenesis (38, 39, 40). Because the product of PI3'-kinase, PtdIns (3, 4, 5) P3, is presumed to function at the downstream signaling, one can speculate that down-regulation of the PI3'-kinase product negatively regulates insulin-induced mitogenic signaling. Recently, SHIP was identified as a member of the inositol 5'-phosphatase family, which hydrolyzes the 5'-phosphate from both inositol- and phosphatidylinositol-phosphatases (12, 13, 14, 15). Because PtdIns (3, 4, 5) P3 is a substrate for SHIP (12, 13, 14, 15), SHIP may regulate PI3'-kinase mediated insulin’s effect by hydrolyzing PtdIns (3, 4, 5) P3 to PtdIns (3, 4) P2. Along this line, expression of SHIP inhibited Xenopus oocytes maturation induced by insulin and a constitutively active PI3'-kinase. Because insulin-induced germinal vesicle breakdown (GVBD) is thought to be mainly mediated by PI3'-kinase in oocytes, the catalytic activity of SHIP appears to have a critical role in regulating insulin-induced GVBD (23). More recently, it has been reported that insulin-induced Glut4 translocation can be negatively regulated by expression of SHIP (41). There is now increasing evidence that SHIP acts as a negative regulator of signaling. Expression of SHIP in myeloid cells led to inhibition of the cell growth (12). SHIP associates with the inhibitory Fc{gamma} receptor IIB subunit (Fc{gamma}RIIB) and is implicated in Fc{gamma}RIIB receptor-mediated negative signaling in B cells and mast cells (20, 21, 22). By targeted disruption of SHIP, numbers of granulocyte-macrophage progenitors were increased as a consequence of hyperresponsiveness to stimulation by macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor, interleukin-3, and steel factor (42). In this regard, our results further extended the notion of negative regulatory role of SHIP during insulin action.

In addition to the negative regulatory role of SHIP presumably through its catalytic activity, SHIP can possibly regulate cell proliferation by modulating the p21ras pathway because the carboxyl-terminus tyrosine phosphorylation sites (917/1020-Tyr) of SHIP can bind to the PTB domain of Shc and the SH2 domain of SHIP can interact with the phosphorylated 317-Tyr residue of Shc in vitro (16, 17). In fact, SHIP could interact with Shc via either SHIP SH2 domain or the carboxyl-terminus tyrosine residues, whereas the interaction via SHIP carboxyl-tyrosines appeared to be relatively greater significance. These results are consistent with the previous reports showing that SHIP interaction via the carboxyl-terminal tyrosines is a predominant mechanism, whereas SHIP SH2 domain also has a role to interact with Shc (16, 17).

Following insulin stimulation, tyrosine-phosphorylated insulin receptor substrates (IRS) and Shc can potentially independently propagate the signal of the activated insulin receptor to Grb2·Sos and stimulation of p21ras-GTP formation (5, 43). However, Shc·Grb2·Sos rather than IRS·Grb2·Sos is shown to be a predominant signaling pathway coupling insulin receptors to p21ras for MAP kinase activation in HIRc cells (5, 43). Our results demonstrated that expression of WT-SHIP inhibited insulin-induced Shc·Grb2 association. The reduction of Shc·Grb2 association was correlated with decreased MAP kinase activity and thymidine incorporation in WT-SHIP cells. In addition, the decrease of insulin-stimulated thymidine incorporation by expression of WT-SHIP was partly compensated by coexpression of Shc. These results indicate that SHIP negatively modulates insulin-induced mitogenesis by apparently regulating Shc·Grb2 pathway in insulin signaling. In addition, expression of the mutant SHIP lacking the SH2 domain only slightly affected insulin-induced Shc·Grb2 association, MAP kinase activation, and thymidine incorporation. Furthermore, in accordance with the results in B65UtA1, DA-3, and Ba/F3 cells (35), we were unable to detect Grb2 in the anti-SHIP immunoprecipitates or SHIP in the anti-Grb2 immunoprecipitates in HIRc cells (data not shown), indicating that individual SHIP·Shc and Grb2·Shc complexes would exist. Although our data are consistent with a notion of competition between SHIP and Grb2 for Shc, we cannot exclude a role for the SH2 domain of SHIP indirectly having an effect on Shc·Grb2 complex. Similarly, we cannot rule out a role for the SHIP-SH2 domain in regulating insulin mediated mitogenesis independent of the effect on Shc·Grb2 complex formation. On the other hand, our results are not in agreement with the report showing that injection of SHIP complementary DNA (cDNA) containing the inactivating SH2 domain mutation functioned almost similarly to inhibit insulin-induced GVBD in oocytes compared with that of wild-type SHIP (23). The different role of the SH2 domain of SHIP may arise from the cells used for these analyses. In this regard, PI3'-kinase plays an important role even in the activation of p21ras-MAP kinase pathway as well as the maturation in oocytes (23). The modulation of Shc·Grb2 pathway for p21ras activation may not have a critical role in the cells. In Rat1 fibroblasts, however, p21ras-MAP kinase pathway is independent of PI3'-kinase pathway (4, 5). Thus, a MEK inhibitor, i.e. PD98059, did not affect insulin-induced PI3'-kinase activity (data not shown). Conversely, pharmacological inhibitor of PI3'-kinase, i.e. wortmannin or LY294002, did not affect insulin stimulation of MAP kinase activity (data not shown). Consequently, insulin signal to Shc·Grb2 pathway could play an important role in p21ras-MAP kinase activation ultimately leading to cell cycle progression in Rat1 fibroblasts.

SHIP becomes tyrosine phosphorylated following activation of the hemopoietic cell surface receptors for numerous cytokines (30, 31, 32, 33, 34). However, it was uncertain whether insulin could induce SHIP phosphorylation. Our results clearly demonstrated that insulin could induce tyrosine phosphorylation of SHIP. Based on the phosphotyrosine binding consensus motif, two tyrosines, Tyr-917 and Tyr-1020, in the carboxyl-terminal region of SHIP were identified as phosphorylation sites for binding to Shc-PTB domain (17). In addition, insulin-induced tyrosine phosphorylation of SHIP is modulated by the SH2 domain of SHIP because the time course of {Delta}SH2-SHIP phosphorylation was somewhat delayed. These results are consistent with the previous findings showing that the SH2 domain is required for SHIP tyrosine phosphorylation in response to IL3 in DA-ER cells (16), and that phosphorylation of carboxyl-tyrosine residues of SHIP is important for SHIP interaction with Shc (17). Thus, the interaction between SHIP and Shc may compete with Shc·insulin receptor association (8, 24). Consequently, expression of SHIP may impose an impact on Shc·Grb2 pathway by affecting tyrosine phosphorylation of Shc. However, it was not the case in Rat1 fibroblasts. Insulin-induced tyrosine phosphorylation of Shc was not affected by expression of either WT-, {Delta}SH2-, or 2F-SHIP. Furthermore, expression of 2F-SHIP as well as WT-SHIP inhibited insulin-induced Shc·Grb2 association and MAP kinase activation. These results indicate that SHIP does not regulate insulin signaling at the level of Shc tyrosine phosphorylation. However, we cannot exclude the possibility that SHIP carboxyl-terminus tyrosines contribute, albeit it is lesser extent, to insulin-induced mitogenesis, because the degree of inhibition in insulin-induced thymidine and BrdU incorporation was slightly less in 2F-SHIP cells than that in WT-SHIP cells as shown in Figs. 7Go and 9Go.

Although our results argue against the principal role of SHIP tyrosine phosphorylation in insulin-induced mitogenic signaling via Shc·Grb2 pathway, one can speculate that SHIP tyrosine phosphorylation may modulate the 5'-phosphatase activity of SHIP, as tyrosine phosphorylation of SHIP was reported to negatively regulate the 5'-phosphatase activity in RBL-2H3 cells (44). Thus, constitutive phosphorylation of SHIP with elevated PtdIns (3, 4, 5) P3 levels is observed in Bcr-Abl-transformed cells (45). Alternatively, it has been shown that tyrosine phosphorylation of SHIP does not affect its 5'-phosphatase in FDC-P1/Fms cells (12) and B6SUtA1 cells (13), whereas it may be required for adequate SHIP localization. Along this line, tyrosine-phosphorylated SHIP can also associate with p85 regulatory subunit of PI3-kinase after cytokine stimulation (46, 47). SHIP has been shown to associate with p85 subunit only via phosphorylated SHIP 917-Tyr residue (47). Therefore, it is possible that SHIP association with Shc and/or p85, via phosphorylated C-terminal tyrosine residue(s), may be important for the adequate localization of SHIP for its functioning. Because the importance of 5'-phosphatase activity of SHIP has been reported by using catalytically inactive SHIP in insulin-induced Xenopus oocyte maturation (23) and Glut4 translocation (41), the role of tyrosine phosphorylation of SHIP in its function related to the regulation of the 5'-phosphatase activity remains to be elucidated.

SHIP is expressed primarily in hematopoietic cells and plays a negative regulatory role (12, 13, 14). In fact, we could only detect a small amount of SHIP in Rat1 fibroblasts. Recently, human SHIP2, which is closely related to SHIP, has been cloned and shown to be more widely expressed in Northern blot analysis (48). Along this line, insulin-induced tyrosine phosphorylation of SHIP2 was reported in 3T3-L1 adipocytes, indicating a role in insulin signaling (49). Because expression of considerable levels of SHIP2 was seen in Rat1 fibroblasts (data not shown), present results with SHIP overexpression might be indication of the role of SHIP2.

In summary, SHIP plays an negative regulatory role in insulin-induced mitogenesis. The SH2 domain of SHIP is important for its negative regulatory function, at least in part, by negatively modulating Shc·Grb2 pathway in Rat1 fibroblasts.


   Acknowledgments
 
We thank Dr. Kodimangalam S. Ravichandran (University of Virginia, Charlottesville, VA) for kindly providing SHIP cDNA and critical comments in the preparation of this manuscript.


   Footnotes
 
1 This work was supported in part by a grant-in-aid for encouragement of young scientists from the Ministry of Education, Science, Sports, and Culture in Japan (to T.S.). Back

Received February 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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