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Molecular Endocrinology, doi:10.1210/me.2004-0096
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Molecular Endocrinology 19 (2): 421-430
Copyright © 2005 by The Endocrine Society

Mechanism of SHIP-Mediated Inhibition of Insulin- and Platelet-Derived Growth Factor-Stimulated Mitogen-Activated Protein Kinase Activity in 3T3-L1 Adipocytes

Prem M. Sharma, Hyun-Shik Son, Satoshi Ugi, William Ricketts and Jerrold M. Olefsky

Department of Medicine (P.M.S., H.-S.S., S.U., J.M.O.), University of California, San Diego, La Jolla, California 92093; and ICN Pharmaceuticals, Inc. (W.R.), Costa Mesa, California 92626

Address all correspondence and requests for reprints to: Prem M. Sharma, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: psharma{at}ucsd.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Src homology 2-containing 5' inositolphosphatases (SHIP and SHIP2) dephosphorylate 3'-phosphorylated PtdIns on the 5' position, decreasing intracellular levels of PtdIns 3,4,5-P3. In the current study, we investigated the role of SHIP in insulin and platelet-derived growth factor (PDGF) signaling by expressing wild-type (WT) and catalytically inactive SHIP{Delta}IP in 3T3-L1 adipocytes, utilizing adenoviral infection. Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and of SHIP{Delta}IP, and tyrosine phosphorylation of SHIP-associated proteins increased after ligand stimulation. Tyrosine-phosphorylated PDGFR, IR, and insulin receptor substrate-1 all immunoprecipitated with SHIP. Expression of WT and {Delta}IP mutant SHIP did not affect tyrosine phosphorylation of either the insulin or the PDGF receptor, or the expression of insulin receptor substrate-1 and Shc proteins. Both SHIP-WT and SHIP{Delta}IP blocked insulin and PDGF-induced MAPK and MAPK kinase phosphorylation as well as, GTP-bound Ras activity, suggesting that the catalytic activity of SHIP is not necessary for these effects. SHIP associated with Shc upon ligand stimulation, indicating that the SHIP-Shc association is phosphorylation dependent. This association was primarily between the SHIP-SH2 domain and the phosphorylated tyrosine residues of Shc because no association was observed when the 3YF-Shc mutant was coexpressed with SHIP. The Shc{bullet}Grb2 association was not compromised by SHIP expression, despite complete inhibition of the Ras/MAPK pathway. Interestingly, son-of-sevenless (SOS) protein normally found in Grb2 complexes was markedly reduced in SHIP expressing cells, whereas the displaced SOS was recovered when the post-Grb2-IP supernatants were blotted with anti-SOS antibody. Thus, SHIP competes son-of-sevenless (SOS) away from Shc-Grb2. In summary, 1) SHIP-WT and SHIP{Delta}IP expression inhibit insulin and PDGF stimulated Ras, MAPK kinase, and MAPK activities; 2) SHIP associates with tyrosine phosphorylated Shc, and the proline-rich sequences in SHIP associate with Grb2 and titrate out SOS to form Shc{bullet}Grb2{bullet}SHIP complexes; and 3) dissociation of SOS from the Shc{bullet}Grb2 complex inhibits Ras GTP loading, leading to decreased signaling through the MAPK pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GROWTH FACTORS SUCH as insulin- and platelet-derived growth factor (PDGF) mediate their cellular responses by binding to and activating cell surface receptors that contain tyrosine kinase domains. After ligand binding, these receptors dimerize, undergo autophosphorylation, and then attract cytoplasmic proteins containing Src homology 2 (SH2), or phosphotyrosine binding (PTB) domains to initiate various intracellular signaling cascades (1, 2). One of the best characterized of these cascades is the Ras pathway (3, 4) and two SH2-containing adapter proteins, Grb2 and Shc, have been implicated in its activation. Specifically, these proteins have been shown to bind either directly to tyrosine-phosphorylated receptors (5, 6) or via docking proteins such as the insulin receptor substrates (IRSs) (7).

Shc, a widely expressed protein that contains an N-terminal phosphotyrosine binding (PTB) domain (8, 9, 10) and a C-terminal SH2 domain (11), can associate, in its tyrosine-phosphorylated form, with the SH2 domain of Grb2. Grb2 exists as a preformed Grb2-son-of-sevenless (SOS) complex, and this interaction may increase further after ligand stimulation (12, 13). Shc binding to Grb2{bullet}SOS complex is thought to be important for the membrane localization of SOS, where it leads to p21Ras activation. Thus, the 25-kDa adapter protein Grb2, with two SH3 domains flanking one SH2 domain, shuttles the Ras guanine nucleotide exchange factor, SOS, to activated receptors or to IRSs so that SOS can activate Ras by catalyzing the exchange of GDP for GTP (5, 7, 12, 14, 15).

Shc has also been shown to associate with SH2 containing 5'-phosphatases, SHIP, and SHIP2, in addition to the interaction with Grb2 (16, 17, 18, 19, 20, 21). Both phosphatases contain different protein-protein interaction domains such as: a N-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, potential phosphotyrosine binding (PTB) consensus sequence(s), NPAY (asparagine-proline-alanine-tyrosine) and a C-terminal proline-rich sequences allowing to bind SH3 domain (17, 18, 19). cDNAs encoding SHIP proteins have been reported in human, mouse, and rat (22, 23, 24). Both SHIP and SHIP2 are transiently tyrosine phosphorylated by growth factor stimulation and by activation of immunoregulatory receptors in various cell models (17, 25, 26, 27).

SHIP functions in part by modifying a signaling pathway that is initiated by activation of phosphatidylinositol 3'-kinase (PI 3-kinase) (28, 29), a lipid kinase with pleiotropic effect (30). SHIP selectively hydrolyzes the 5'-phosphate from inositol 1,3,4,5-tetraphosphate (Ins 1,3,4,5-P4) and phosphatidylinositol 3,4,5-triphosphate (PtdIns 3,4,5 P3), the latter being a product of PI 3-kinase activity (16, 17, 18, 19, 20, 21, 22, 31). However, it is not entirely clear at this time how such changes in phosphatidylinositol metabolism mediate biological effects. Mice with a disruption of the SHIP gene fail to thrive and develop a myeloproliferative disorder with extensive infiltration of myeloid cells in the lung (32). SHIP2 has been shown to control insulin sensitivity in a model of SHIP2-deficient mice (33). In cell line models, SHIP negatively regulates growth, differentiation, or migration, and it may have an important role in apoptosis (19, 21, 34, 35). The enzyme activity of SHIP has not been shown to change after receptor activation, suggesting that relocation of SHIP to the cell membrane may be critical for signaling (36). Therefore, transient interaction of SHIP with signaling complexes associated with transmembrane receptors or membrane associated proteins is likely to be important in regulating its function.

Given the potential role of SHIP in regulation of PI 3-kinase signaling by growth factors and insulin (26), we aimed to study the effect of expressing SHIP and a catalytically inactive mutant of SHIP (SHIP{Delta}IP) on insulin- and PDGF-induced Ras/MAPK pathway in 3T3-L1 adipocytes. In the course of these studies, we observed that SHIP inhibits insulin and PDGF-stimulated Ras and MAPK activation but did not have a negative effect on ligand-stimulated Shc{bullet}Grb2 association. We also observed that SHIP associated with Shc only upon ligand stimulation and that the phosphorylated tyrosine residues within the Shc CH domain are necessary for Shc{bullet}SHIP interaction. SHIP was, however, constitutively associated with Grb2, whereas SOS levels were significantly low in this complex. Thus, the SHIP{bullet}Grb2 complex competes SOS away from Shc{bullet}Grb2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Adenovirus-Mediated Expression of SHIP in 3T3-L1 Adipocytes
SHIP is a 5' phosphatase that converts PtdIns 3,4,5 P3 (PIP3) to PtdIns 3,4 P2 (17, 18, 19). To assess the role of SHIP and PIP3 in insulin and PDGF-induced signaling, we expressed HA (hemagglutinin)-tagged wild-type (SHIP-WT) and a phosphatase inactive mutant of SHIP (SHIP{Delta}IP) in 3T3-L1 adipocytes (Fig. 1AGo). The cells were infected with 20 and 100 multiplicity of infection (m.o.i.) of each virus for 16 h, and the proteins were then allowed to express for 48 h. Expression of proteins was confirmed by immunoblotting using an anti-SHIP antibody. As seen in Fig. 1BGo, specific bands appeared in a dose-dependent manner at approximately 145 kDa corresponding to SHIP.



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  		Fig. 1. Structure, Expression, and Tyrosine Phosphorylation of SHIP Constructs

A, Structure of SHIP-WT and SHIP{Delta}IP are shown. The three domains of SHIP are an SH2 domain, a 5'-phosphatase domain, and a carboxyl-terminal proline-rich domain containing tyrosine phosphorylation sites. B, Expression of SHIP in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were uninfected (lanes 1 and 4) or infected with recombinant adenoviruses expressing the SHIP-WT and SHIP{Delta}IP (lanes 2 and 3 and 5 and 6) proteins at 20 and 100 m.o.i. for 16 h and total cell lysates were prepared 48 h later, as described in Materials and Methods. Equal amounts of protein (~30 µg) were resolved by SDS-PAGE, electrophoretically transferred to nitrocellulose membrane, and blotted with anti-SHIP antibody. Immunoreactive bands were detected by enhanced chemiluminescence for the corresponding proteins as indicated. C, SHIP-WT and SHIP{Delta}IP are tyrosine phosphorylated by insulin and PDGF. Differentiated 3T3-L1 adipocytes were uninfected or infected with Ad-Ctrl (empty vector), SHIP-WT and SHIP{Delta}IP adenoviruses at 60 m.o.i for 16 h. Forty-eight hours post infection, cells were serum starved for 16 h and stimulated with insulin (100 ng/ml) or PDGF (50 ng/ml) for 5 min. Whole cell lysates from each group were immunoprecipitated (~300 µg protein per lane) with anti-HA (SHIP) antibody. The immune complexes were subjected to SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and SHIP{Delta}IP. Tyrosine-phosphorylated PDGFR, IR, and IRS-1 all immunoprecipitated with SHIP. The experiment was repeated several times.

 
Effect of SHIP on Insulin and PDGF Receptor Phosphorylation
To assess the effect of insulin and PDGF on SHIP tyrosine phosphorylation as well as the effect of SHIP expression on PDGFR and IR phosphorylation, differentiated 3T3-L1 adipocytes were infected either with control (empty vector), SHIP-WT or SHIP{Delta}IP adenoviruses. Cells were stimulated with either ligand (5 min), lysed, and SHIP was immunoprecipitated with anti-HA antibody, followed by blotting with antiphosphotyrosine antibody (Fig. 1CGo). Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and of SHIP{Delta}IP. Tyrosine-phosphorylated PDGFR, IR, and IRS-1 all immunoprecipitated with SHIP (Fig. 1CGo), and tyrosine phosphorylation of the SHIP associated proteins clearly increased after ligand stimulation. Expression of SHIP proteins did not affect the magnitude of PDGF- and insulin-induced tyrosine phosphorylation of their respective receptors or of IRS-1, compared with cells expressing a control plasmid (data not shown).

SHIP Attenuates MAPK Kinase (MEK) and MAPK Phosphorylation
To assess the effect of SHIP on mitogenic signaling, we measured insulin and PDGF stimulated MEK and MAPK activation using phospho-specific MAPK (Fig. 2AGo, upper panel) or MEK antibodies (Fig. 2BGo, upper panel). Both insulin and PDGF stimulated MAPK and MEK phosphorylation, which was attenuated by SHIP-WT and SHIP{Delta}IP. The expression levels of ERK1/2 and MEK proteins were not altered by SHIP-WT and SHIP{Delta}IP (Fig. 2Go, A and B, lower panels).



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  		Fig. 2. SHIP-WT and SHIP{Delta}IP Attenuate Insulin- and PDGF-Stimulated MAPK and MEK Phosphorylation in 3T3-L1 Adipocytes

The differentiated 3T3-L1 adipocytes were infected with Ctrl (lanes 1–3), SHIP-WT (lanes 4–6), or SHIP{Delta}IP (lanes 7–9) adenoviruses at 60 m.o.i. for 16 h. After 48 h, the cells were starved for 16 h and stimulated with 100 ng/ml insulin or PDGF (50 ng/ml) for 5 min. Whole cell lysates were prepared and analyzed by Western blotting using phospho-MAPK antibody (A, upper panel) or phospho-MEK antibody (B, upper panel). The same cell lysates were analyzed by Western blotting using anti-ERK-1 antibody (A, lower panel) and anti-MEK antibody (B, lower panel). C, HIRcB fibroblasts were cotransfected with HA-ERK, SHIP-WT or SHIP{Delta}IP transiently with SuperFECT. After transfection, cells were allowed to grow for 24 h before being serum starved for 16 h followed by insulin (100 ng/ml) stimulation for 5 min. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with phosphospecific MEK antibody. The same cell lysates were analyzed by Western blotting using anti-ERK antibody (C, lower panel). The experiment was repeated three times with similar results.

 
These results were reconfirmed in HIRcB fibroblasts transiently cotransfected with HA-ERK and SHIP-WT or SHIP{Delta}IP. Cell lysates from insulin-stimulated cells were immunoprecipitated with anti-HA antibody and immunoblotted with phospho-specific MEK antibody. Insulin-stimulated phosphorylation of p42MAPK was significantly inhibited by SHIP-WT and SHIP{Delta}IP, with no change in ERK protein expression (Fig. 2CGo).

Because SHIP-WT and SHIP{Delta}IP led to similar findings, we conclude that the catalytic activity of SHIP is not necessary for these effects.

SHIP Inhibits Insulin- and PDGF-Stimulated Ras Activity
To assess Ras activation, we used the specificity of the binding interaction between Ras-GTP and the Ras binding domain (RBD) of Raf-1, which provides a measure of the amount of GTP bound Ras (37, 38). As seen in Fig. 3Go, insulin and PDGF stimulation led to approximately 5- and 8-fold increases in Ras activity, respectively. Both the WT and phosphatase inactive SHIP completely extinguished insulin and PDGF-stimulated Ras activity (Fig. 3Go), with no change in Ras protein expression.



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  		Fig. 3. SHIP-WT and SHIP{Delta}IP Attenuate Insulin- and PDGF-Stimulated Ras Activation in 3T3-L1 Adipocytes

The differentiated 3T3-L1 adipocytes were infected with Ctrl (lanes 1–3), SHIP-WT (lanes 4–6) or SHIP{Delta}IP (lanes 7–9) at 60 m.o.i. for 16 h. After 48 h, the cells were starved for 16 h and stimulated with 100 ng/ml insulin or PDGF (50 ng/ml) for 5 min. Whole cell lysates were prepared and precipitated with the GST-RBD fusion protein. The precipitates were then analyzed by Western blotting using anti-Ras antibody as described in Materials and Methods (upper panel). To verify equal expression of Ras protein, cell lysates were immunoblotted with anti-Ras antibody (lower panel).

 
Effect of SHIP Expression on Insulin- and PDGF-Induced Shc Association with Grb2 and SHIP
Tyrosine-phosphorylated Shc functions as an upstream activator of Ras by recruiting the Grb2.SOS complex to the plasma membrane where SOS mediates Ras-GTP loading. We assessed the functions of these complexes in the presence of SHIP or SHIP{Delta}IP expression. For these experiments, Shc was immunoprecipitated with an anti-Shc antibody and immunoprecipitates were immunoblotted with antiphosphotyrosine antibody. After insulin and PDGF stimulation (5 min), Shc was tyrosine phosphorylated, and this was not affected by either SHIP-WT or SHIP{Delta}IP, compared with control infected cells (Fig. 4AGo, middle panel). We next determined the effect of SHIP-WT and SHIP{Delta}IP expression on the association of Shc with SHIP and Grb2. Thus, equal fractions of the Shc immunoprecipitates prepared above were blotted with anti-SHIP or anti-Grb2 antibodies. In the basal state, we found a minimal association of Shc with Grb2 and none with SHIP. After ligand stimulation, Shc association with both SHIP and Grb2 increased significantly (Fig. 4AGo, upper and lower panels), and this effect was greater in the SHIP and SHIP{Delta}IP cells. Expression of Shc protein in whole cell lysates from each group was comparable (data not shown).



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  		Fig. 4. Effect of SHIP Expression on Insulin- and PDGF-Induced Shc Association with Grb2 and SHIP in 3T3-L1 Adipocytes

The differentiated 3T3-L1 adipocytes were infected with Ctrl (lanes 1–3), SHIP-WT (lanes 4–6) or SHIP{Delta}IP (lanes 7–9) at 60 m.o.i. for 16 h. After 48 h, the cells were starved for 16 h and stimulated with 100 ng/ml insulin or PDGF (50 ng/ml) for 5 min. Whole cell lysates were prepared and immunoprecipitated with anti-Shc antibody, followed by immunoblotting with anti-SHIP antibody (A, upper panel), antiphosphotyrosine antibody (middle panel), and anti-Grb2 antibody (lower panel). The experiment was repeated three times with similar results. B, Effect of SHIP expression on insulin- and PDGF-induced Grb2 association with Shc, SHIP, and SOS in 3T3-L1 adipocytes. Whole cell lysates were prepared as described in Fig. 4AGo and were immunoprecipitated with anti-Grb2 antibody, followed by immunoblotting with anti-SOS antibody (upper panel), anti-SHIP antibody (third panel), and anti-Shc antibody (lower panel). The post-Grb2 IP supernatants were immunoblotted with anti-SOS antibody (second panel). The experiment was repeated three times and representative blots are shown. C, Expression of SHIP2-WT and tSHIP/{Delta}pro in HIRcB fibroblasts. HIRcB fibroblasts were transfected with His6-tagged vector alone (lanes 1 and 2), SHIP2-WT (lanes 3

and 4) or tSHIP/{Delta}pro (lanes 5 and 6). Twenty-four hours post transfection, cells were serum starved for 16 h and stimulated with 100 ng/ml insulin (lanes 2, 4, and 6) for 5 min. Whole cell lysates were prepared and analyzed by Western blotting using anti-His6 antibody to verify equivalent expression of both proteins. The experiment was repeated three times with similar results. D, Modulation of insulin-induced Grb2 association with SOS by tSHIP/{Delta}pro expression in HIRcB fibroblasts. Whole cell lysates were prepared as described in Fig. 4CGo, and were immunoprecipitated with anti-Grb2 antibody, followed by immunoblotting with anti-SOS antibody. The experiment was repeated three times.

 
Effect of SHIP Expression on Insulin- and PDGF-Induced Grb2 Association with Shc, SHIP, and SOS
The adapter protein Grb2 consists of one SH2 domain flanked by two SH3 domains. The Grb2 SH2 domain binds to the phosphorylated Tyr-317 residue of Shc, and the SH3 domain interacts with the proline-rich sequences in the C terminus of the guanine nucleotide exchange factor-SOS. The proline-rich sequence within the C terminus of SHIP suggests a possible interaction of this protein with Grb2. Thus, SHIP may compete with SOS for Grb2 binding. Therefore, we examined the effect of SHIP expression on insulin- and PDGF-induced association of Grb2 with Shc, SHIP, and SOS.

Cell lysates from basal and ligand-stimulated cells were immunoprecipitated with anti-Grb2 antibody, and immunoblotted with anti-Shc, anti-SHIP, and anti-SOS antibodies. In Grb2 immunoprecipitates (Fig. 4BGo), a ligand-induced increase in Shc association was observed, which was somewhat increased in the SHIP and SHIP{Delta}IP cells, consistent with Fig. 4AGo. Grb2 associated constitutively with SHIP under all conditions, presumably reflecting interactions between the Grb2 SH3 domain and the polyproline region of SHIP. Importantly, SHIP and SHIP{Delta}IP expression almost completely inhibited the association of SOS with Grb2. Taken together, these results indicate that SHIP can interact directly with tyrosine-phosphorylated Shc through its SH2 domain and with the Grb2 SH3 through its proline-rich region and that this allows SHIP to displace SOS from the Shc{bullet}Grb2 complex, which effectively blocks ligand mediated Ras activation.

To further confirm this idea, we immunoblotted the post-Grb2 IP supernatants and found a markedly increased SOS content after SHIP WT and SHIP{Delta}IP expression, showing that the SOS displaced from the Grb2{bullet}SOS complex by SHIP protein could be recovered in the unbound state.

Along similar lines, we assessed the role of the SHIP proline-rich domain in interrupting the Grb2{bullet}SOS complex by using a mutant SHIP construct in which the C-terminal proline-rich region was deleted (tSHIP/{Delta}pro) (39). Comparable amounts of SHIP WT and tSHIP/{Delta}pro were expressed in HIRcB cells followed by insulin stimulation and Grb2 antibody immunoprecipitation (Fig. 4Go, C and D). In agreement with the results in Fig. 4BGo, SHIP2 expression inhibited Grb2 association with SOS by about 50%. However, in the tSHIP/{Delta}pro expressing cells, SOS association with Grb2 was only minimally affected (about 10% decrease), indicating that SHIP2 interacts with Grb2 and displaces it from the Grb2{bullet}SOS complex, at least in part, through its proline-rich region.

Mechanism of Shc{bullet}SHIP Association
To more specifically assess the association of Shc with SHIP, we constructed FLAG-tagged, WT Shc, and mutant Shc proteins containing: 1) 3YF Shc in which Tyr 239, 240, and 317 are mutated to phenylalanine, and 2) R401L Shc, an SH2 domain mutant in which arginine at residue 401 within the conserved FLVR motif is mutated to lysine (Fig. 5AGo). These constructs were transiently coexpressed along with HA-tagged SHIP into HIRcB cells (Fig. 5BGo). SHIP was immunoprecipitated with epitope-specific HA antibody, and its association with Shc proteins in response to insulin and epidermal growth factor (EGF) stimulation was analyzed by Western blotting using anti-FLAG antibody. For these experiments, EGF was used as the ligand instead of PDGF because these cells contain very few PDGF receptors. As shown in Fig. 5BGo, WT Shc and R401L Shc, both associated with SHIP upon ligand stimulation. However, Y3F Shc failed to associate with SHIP, with or without insulin or EGF stimulation. To ensure equal SHIP immunoprecipitation, the anti-HA immunoprecipitates were also immunoblotted with anti-HA antibody.



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  		Fig. 5. Effect of SHIP on Insulin- and EGF-Stimulated As-sociation with WT and Various Mutants of Shc in HIRcB Fibroblasts

A, A schematic diagram showing various constructs of flag-tagged Shc cotransfected with HA-tagged SHIP. WT Shc contains the full-length protein, the 3YF is a triple tyrosine mutant of the CH domain in which the three tyrosine residues at position 239, 240, and 317 are mutated to phenylalanine, and the SH2 domain mutant (R401L) contains an argenine at residue 401 within the conserved FLVR motif mutated to a lysine. B, HIRcB fibroblasts were cotransfected with HA-tagged SHIP-WT transiently with flag-tagged Shc wild-type, WT (lanes 1–3), and mutant Shc proteins: 3YF (lanes 4–6), R401L (lanes 7–9), as described in Materials and Methods. Cells were serum starved and stimulated with 100 ng/ml insulin (lanes 2, 5, and 8) or 10 ng/ml EGF for 5 min. Whole cell lysates were prepared and SHIP was immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG antibody to determine coprecipitated Shc (top panel). The levels of transduced SHIP protein was determined by immunoblotting approximately 2% of the HA-immunoprecipitates with anti-HA antibody (middle panel), and that of Shc was determined by immunoblotting cell lysates with anti-FLAG antibody (lower panel).

 
These results further demonstrate that the SHIP{bullet}Shc association is ligand dependent and that the phosphorylated tyrosine residues within the Shc CH domain are necessary for the Shc{bullet}SHIP interaction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SHIP is a member of the inositol 5'-phosphatase family, which hydrolyzes the 5'-phosphate from both inositol- and phosphatidylinositol-phosphates. It has been demonstrated that SHIP is a negative regulator of insulin-stimulated MAPK (40), and in the current study, we have explored the mechanisms underlying the effect of SHIP to inhibit insulin and PDGF signaling through the Ras/MAPK pathway. Our results indicate that SHIP forms a trimeric complex through its interactions with both Shc and Grb2, and that the proline-rich regions of SHIP displaces SOS from the Shc{bullet}Grb2 complex, leading to inhibition of Ras activation with decreased signaling to MAPK.

SHIP contains several recognizable protein/protein interaction motifs and SHIP function depends on its association with upstream proteins as well as downstream effector molecules. To investigate the mechanisms of SHIP’s negative effects on MAPK signaling, we overexpressed SHIP-WT and SHIP{Delta} IP, using adenoviral gene transfer in 3T3-L1 adipocytes. Both SHIP proteins effectively quenched insulin- and PDGF- stimulated MAPK activity and, because SHIP{Delta} IP lacks the 5'-phosphatase activity, these results indicate that the inhibitory effects of SHIP on MAPK signaling are not mediated through its catalytic function.

Insulin and other growth factors are known to stimulate tyrosine phosphorylation of SHIP at positions 917 and 1020 (26, 41, 42, 43), and because SHIP can be recruited to the IL4, EGF, and erythropoeitin receptors in an SH2-dependent manner (27, 39, 44), we determined whether such interactions could also be observed with SHIP and the insulin and PDGF receptors. We found that insulin and PDGF lead to tyrosine phosphorylation of SHIP and that insulin and PDGF receptors can then coimmunoprecipitate with SHIP. In analogy to the EGF receptor interaction (39), these data indicate that the SH2 domain of SHIP binds to tyrosine-phosphorylated sites on these activated receptors. In turn, the tyrosine-phosphorylated residues of SHIP create docking sites for SH2 and PTB domain containing proteins and, in our studies, we find that tyrosine-phosphorylated SHIP interacts with both Shc and IRS-1 proteins in response to either insulin or PDGF stimulation.

Overexpression of either SHIP-WT or SHIP{Delta} IP inhibits insulin- and PGDF-stimulated Ras and MEK activation, indicating that the effects of SHIP to block MAPK signaling are exerted proximal to Ras activation. Furthermore, SHIP proteins did not have any effect on phosphorylation of the insulin or PDGF receptors, or on ligand-mediated tyrosine phosphorylation of the receptor substrates, IRS-1 and Shc. Taken together, these results indicate that the site of inhibition of MAPK signaling by SHIP is proximal to Ras and distal to the receptor substrates IRS-1 and Shc. PGDF signals to the Ras/MAPK pathway by enhancing Shc tyrosine phosphorylation, which then engages the Grb2{bullet}SOS complex leading to Ras activation. Although insulin can potentially lead to activation of the Ras/MAPK pathway by stimulating phosphorylation of IRS-1 or Shc, previous studies have indicated that signaling through Shc is the dominant pathway coupling the insulin receptor to p21 Ras activation (45). Therefore, because the inhibitory effects of SHIP are exerted proximal to Ras and distal to the receptor substrates, we focused our attention on the interactions of SHIP with the Shc/Grb2/SOS complex.

Shc is tyrosine phosphorylated at positions 239, 240, and 317 in response to either insulin or PGDF, and we found that after ligand stimulation, the association of SHIP with Shc is markedly enhanced. Because both proteins are tyrosine phosphorylated, and both have SH2 domains, with Shc also having a PTB domain, multiple potential mechanisms of interaction exist. To assess this, we evaluated the interaction of SHIP with mutant Shc proteins. When all three tyrosines within the CH domain of Shc were mutated to phenylalanine (3YF Shc), the mutant Shc protein was unable to associate with SHIP in response to either insulin or EGF. In contrast, when the Shc SH2 domain was disabled (R401L), association with SHIP was unaffected. These data indicate that the primary mechanism of interaction between Shc and SHIP occurs between the SH2 domain of SHIP and tyrosine residue of Shc. These results are consistent with previous studies that showed that SHIP proteins containing mutated SH2 domains are incapable of interacting with Shc in a murine hematopoietic cell line (21). Similarly, glutathione-S-transferase (GST) fusion proteins containing the SHIP SH2 domain can directly precipitate tyrosine phosphorylated Shc in cell-free experiments (21, 46). On the other hand, other mechanisms may also exist in different cell types because an earlier report showed that phosphorylation of carboxy-terminal tyrosine residues of SHIP are important for interactions with Shc (20).

The reliance of the SHIP{bullet}Shc interaction on Shc tyrosine phosphorylation indicates that inhibition of Shc tyrosine phosphorylation would not be a mechanism for SHIP’s negative effects on Ras/MAPK activation. Indeed, we show that overexpression of SHIP has no effect on Shc phosphorylation. These data suggest that some aspect of the SHIP{bullet}Shc interaction provides the inhibitory signal to downstream Ras/MAPK signaling. Indeed, the data in Fig. 4Go provide a novel mechanism for this effect. Thus, we find that in the basal, unstimulated state, Grb2 is constitutively associated in a complex with SOS, and this is well known in the literature. Upon ligand stimulation with either insulin or PGDF, SHIP associates with Shc and Grb2. Surprisingly, SOS is no longer detected in this complex but was now detectable and fully recovered in the post-Grb2 IP supernatants. Because SHIP can associate with Grb2 in the basal state, and because insulin and PDGF both stimulate Shc tyrosine phosphorylation, we propose that binding of SHIP to the Grb2{bullet}Shc complex interferes with the SOS{bullet}Grb2 interaction effectively competing SOS away from this signaling complex. Because SOS is the upstream activator of Ras, this effectively inhibits Ras activation with decreased downstream signaling to MAPK. It is probable that the proline-rich domains of SHIP interact with the SH3 domains of Grb2, inhibiting SOS association with these same SH3 domains. This scenario is consistent with work by Kavanaugh et al. (18), who initially identified SHIP using the strategy based on expression cloning via Grb2 binding. Indeed, Kavanaugh et al. (18) have demonstrated that binding of SHIP to Grb2 requires both SH3 domains of Grb2, but not the SH2 domain. Damen et al. (17) also show that the Grb2 N-terminal SH3 domain physically interacts with SHIP.

Finally, membrane localization of cytosolic protein is a common means by which enzymes involved in signaling pathways carry out their functions. Along these lines, it has been shown that SHIP phosphorylation and receptor tyrosine engagement has only a minimal effect on SHIP’s enzymatic activity. However, redistribution of SHIP to the plasma membrane upon ligand stimulation resulted in increased enzymatic activity (36). Shc and Grb2 are known to interact with a variety of membrane-bound proteins and phosphoinositides. Therefore, SHIP may employ interactions via these adapters to become localized to the cell membrane, where the substrates for its catalytic activity reside.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Porcine insulin was kindly provided by Eli Lilly (Indianapolis, IN). PDGF was from Invitrogen Life Technologies (Gaithersburg, MD). Phospho-specific MAPK antibody, phospho-specific MEK antibody, and MEK antibody were from New England Biolabs (Beverly, MA). Polyclonal anti-Shc antibody, monoclonal anti-Grb2 antibody, and antiphosphotyrosine antibody (4G10) were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-His6 antibody was purchased from Calbiochem (San Diego, CA). Anti-HA antibody (12CA5) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Anti-FLAG antibody, anti-SOS antibody, anti-SHIP-1 antibody, polyclonal anti-Grb2 antibody, horseradish peroxidase-linked antirabbit, antimouse, and antigoat antibodies, and protein G-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ras antibody, and monoclonal anti-Shc antibody were from Transduction Laboratories (Lexington, KY). DMEM and fetal calf serum were obtained from Life Technologies (Grand Island, NY). XAR-5 film was obtained from Eastman-Kodak (Rochester, NY). All other reagents and chemicals were purchased from Sigma (St. Louis, MO).

Cell Culture
3T3-L1 cells were maintained in DMEM-high glucose (Invitrogen Life Technologies) supplemented with 10% FCS and Penicillin G/Streptomycin (Omega Scientific, Tarzana, CA) and differentiated into adipocytes as previously described (47). Before experiment, the adipocytes were trypsinized and reseeded in the appropriate culture dishes.

Rat-1 fibroblasts overexpressing wild-type human insulin receptors (HIRc-B) were maintained in DMEM-Ham’s F-12 (Invitrogen Life Technologies) supplemented with 10% fetal calf serum and gentamycin (Gemini Bioproducts, Calabasas, CA), 2 mM Glutamax (Invitrogen Life Technologies), and 500 nM methotrexate (Sigma). The Ad-EIA-transformed human embryonic kidney cell line 293 cells were cultured as described previously (48, 49).

SHIP Expression Plasmids
The cDNA for SHIP was cloned into a pCGN vector that contains a CMV promoter and has been described elsewhere (50). It has been modified to contain an HA sequence at its NH2 terminus. Catalytically inactive SHIP was generated by deleting amino acids residues 666–680 (NLPSWCDRVLWKSYP) within the presumed inositol phosphatase catalytic domain (SHIP{Delta}IP), and has been described previously (50).

A truncated form of SHIP2 of 874 amino acids that lacks 366 amino acids containing the proline-rich region at the C terminus, and referred to as tSHIP/{Delta}pro was generated by PCR from the WT SHIP2 (39). The WT and tSHIP/{Delta}pro were subcloned into pcDNA3-His C (Invitrogen Life Technologies). These constructs were transfected into HIRcB fibroblasts with the Fugene transfection reagent (QIAGEN, Valencia, CA).

Preparation of Recombinant Adenovirus
The recombinant adenovirus containing the cDNA encoding the WT SHIP and SHIP{Delta}IP lacking amino acids 666–680 were isolated by homologous recombination with two plasmids, pACCMVpLpA and pJM17, described previously (47, 48, 49). The recombinant plasmids, pAC-SHIP-WT, pAC-SHIP{Delta}IP, and pJM17 were purified and cotransfected into 293 cells. Because 293 cells were originally derived from adenovirus transformation, the missing E1 gene function of pJM17 was provided in trans. The resulting recombinant adenoviruses containing the SHIP-WT, SHIP{Delta}IP were denoted as Ad5-SHIP-WT and Ad5-SHIP{Delta}IP, and were replication defective (at least in cells lacking the E1 region of adenovirus) but are fully infectious.

Cell Treatment
3T3-L1 adipocytes were transduced at a m.o.i. of 20–100 plaque-forming units/cell for 16 h with the recombinant adenoviruses, Ad5-SHIP-WT and Ad5-SHIP{Delta}IP. Transduced cells were incubated for 60 h at 37 C under 10% CO2 in DMEM high-glucose medium with 2% heat-inactivated serum, followed by starvation for 18 h. The efficiency of adenovirus-mediated gene transfer was approximately 90% as measured by immunocytochemistry. The survival of the differentiated 3T3-L1 adipocytes was unaffected by incubation of cells with the different adenovirus constructs because the total cell protein remained the same in infected or uninfected cells.

Preparation of Whole Cell Lysates and Immunoprecipitation
Starved cells were stimulated with ligands at 37 C and lysed in solubilizing buffer [20 mM Tris (pH 7.5), 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 50 mM sodium fluoride, 50 U aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride], for 30 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. For immunoprecipitaton, cell lysates were incubated with primary antibody for 6 h at 4 C and protein A/G-agarose for an additional 2 h. The immunoprecipitates were washed three times with solubilizing buffer, resuspended in Laemmli sample buffer containing 100 mM dithiothreitol, and heated at 100 C for 5 min.

Immunobloting
Whole cell lysates and antibody immunoprecipitates were resolved by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-P; Bedford, MA). Membranes were blocked and probed with specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer’s instructions (Pierce, Rockford, IL).

Ras Activation Assay
Ras activity was determined by the method of Taylor and Shalloway (38). A cDNA encoding for a GST-RBD fusion protein was kindly provided by Dr. Jeffrey E. Pessin (University of Iowa, Iowa City, IA). Briefly, infected or uninfected cells were starved for 16 h, stimulated with 100 ng/ml insulin or 50 ng/ml PDGF for 5 min at 37 C and lysed in lysis buffer [25 mM HEPES (pH 7.5), 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.25% deoxycholate, 25 mM sodium fluoride, 10 mM magnesium chloride, 1 mM sodium vanadate, 1 µg/ml leupeptin, 50 U aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride]. Clarified supernatants were incubated with the GST-RBD bound to glutathione-sepharose beads (20 µg, Amersham Pharmacia Biotech) for 1 h at 4 C. The sepharose beads were washed three times with lysis buffer and analyzed by Western blotting using anti-Ras antibody.

Transient Transfection of HIRcB Cells
The FLAG-tagged Shc expression vector, pRK5 Shc was a generous gift from Dr. Edward Y. Skolnik (Skirball Institute, New York, NY). Mutant Shc cDNAs (Tyr 239, 240, and 317-phenylalanine: 3YF and Arg 401-Lys: R401L) were generated by PCR with a mutagenic oligonucleotide and subcloned into pRK5 as previously described (51). Transient transfection into HIRcB cells was performed with SuperFECT or Fugene reagents (QIAGEN) in accordance with manufacturer’s instructions. After transfection, cells were allowed to grow for 16 h before being serum starved for 24 h before experiments as previously described (51).


   ACKNOWLEDGMENTS
 
We thank Jeffrey E. Pessin, University of Iowa, for providing the GST-RBD. We are grateful to Christophe Erneux of Interdisciplinary Research Institute, Université Libre de Brussels, Belgium, for providing the SHIP2 and tSHIP/{Delta}pro plasmids. We also thank Donna Reichart and Jay Nelson for providing differentiated 3T3-L1 adipocytes, and Elizabeth Hansen for editorial assistance.


   FOOTNOTES
 
This work was supported in part by National Institutes of Health (NIH) Grant DK-33651 (to J.M.O.). P.M.S. is the recipient of the NIH 21 Award DK-067411-01.

First Published Online October 14, 2004

Abbreviations: EGF, Epidermal growth factor; FCS, fetal calf serum; GST, glutathione-S-transferase; HA, hemagglutinin; HIRc, Rat1 fibroblasts overexpressing the human insulin receptor; IRS, insulin receptor substrate; MEK, MAPK kinase; m.o.i., multiplicity of infection; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; PIP3, 3,4,5 phosphatidylinositol; PTB, phosphotyrosine binding; PtdIns, phosphoinositides; SH2, Src homology 2; SHIP, SH2 domain-containing 5' inositolphosphatase; SOS, son-of-sevenless; WT, wild-type.

Received for publication March 5, 2004. Accepted for publication October 5, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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