Membrane Localization of Src Homology 2-Containing Inositol 5'-Phosphatase 2 via Shc Association Is Required for the Negative Regulation of Insulin Signaling in Rat1 Fibroblasts Overexpressing Insulin Receptors
Hajime Ishihara,
Toshiyasu Sasaoka,
Manabu Ishiki,
Tsutomu Wada,
Hiroyuki Hori,
Syota Kagawa and
Masashi Kobayashi
First Department of Internal Medicine (H.I., M.I., T.W., H.H.,M.K.) and the Department of Clinical Pharmacology (T.S., S.K.), Toyama Medical & Pharmaceutical University, Toyama 930-0194, Japan; and the Sainou Hospital (H.I.), Toyama 930-0887, Japan
Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, Toyama Medical& Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: tsasaoka-tym{at}umin.ac.jp.
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ABSTRACT
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Lipid phosphatase SHIP2 [Src homology 2 (SH2)-containing inositol 5'-phosphatase 2] has been shown to be a physiologically critical negative regulator of insulin signaling. We investigated the molecular mechanism by which SHIP2 negatively regulates insulin-induced phosphorylation of Akt, a key downstream molecule of phosphatidylinositol 3-kinase important for the biological action of insulin. Overexpression of wild-type SHIP2 (WT-SHIP2) inhibited insulin-induced phosphorylation of Akt at both Thr308 and Ser473 in Rat1 fibroblasts expressing insulin receptors. The degree of inhibition was less in the cells expressing either a mutant SHIP2 with R47Q change (R/Q-SHIP2) in the SH2 domain, or a mutant SHIP2 with Y987F change (Y/F-SHIP2) in the C-terminal tyrosine phosphorylation site. However, on addition of a myristoylation signal, WT-SHIP2, R/Q-SHIP2, and Y/F-SHIP2 all efficiently inhibited insulin-induced Akt phosphorylation at both residues, whereas a 5'-phosphatase-defective mutant SHIP2 (
IP-SHIP2) with the myristoylation signal did not. Interestingly, the degree of inhibition of Akt phosphorylation by R/Q-SHIP2 and Y/F-SHIP2 is well correlated with the extent of their association with Shc. In addition, overexpression of WT-Shc increased the insulin-induced association of SHIP2 with Shc, whereas a decrease in the amount of Shc on expression of antisense Shc mRNA led to a reduction in the SHIP2-Shc association. Furthermore, the inhibitory effect on insulin-induced Akt phosphorylation by WT-SHIP2 was decreased in antisense-Shc cells. These results indicate that the membrane localization of SHIP2 with its 5'-phosphatase activity is required for negative regulation of insulin-induced Akt phosphorylation and that the localization is regulated, at least in part, by the association of SHIP2 with Shc in Rat1 fibroblasts.
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INTRODUCTION
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THE ACTIVATED INSULIN receptor phosphorylates adaptor proteins such as the insulin receptor substrate family and Shc on tyrosine residues(1, 2, 3). Insulin receptor substrate proteins propagate insulin signals to the p85 regulatory subunit of phosphatidylinositol (PI)3-kinase, activating its p110 catalytic subunit. Insulin-induced PI3-kinase activation is shown to be extremely important for the subsequent performance of a variety of insulin-induced metabolic actions including glucose uptake and glycogen synthesis (1, 2, 3). PI3-kinase functions as a lipid kinase which phosphorylates the D-3 position of PI, PI4-phosphate [PI (4)P], and PI4,5-bisphosphate [PI(4,5)P2] in vitro, whereas in vivo PI3-kinase preferentially phosphorylates PI(4,5)P2 to produce PI3,4,5,-triphosphate [PI(3,4,5)P3] (4). PI(3,4,5)P3 can be subsequently hydrolyzed to PI3,4-bisphosphate [PI(3,4)P2] by the phosphoinositol 5'-phosphatase (5). Thus, insulin treatment increases the cellular amount of PI(3,4,5)P3 and PI(3,4)P2, which can possibly serve as lipid second messengers to relay the signal to downstream target molecules of PI3-kinase resulting in insulins metabolic action. Src homology 2 (SH2)-containing inositol 5'-phosphatase 1 (SHIP1) is known to be such a candidate possessing 5'-phosphatase activity for the hydrolysis of PI(3,4,5)P3 to PI(3,4)P2. Exogenous expression of SHIP1, but not 5'-phosphatase-defective SHIP1, inhibited insulin-induced Xenopus oocyte maturation and insulin stimulation of Glut4 translocation in 3T3-L1 adipocytes (6, 7). Despite these facts, the expression of SHIP1 is relatively restricted to hematopoietic cells, and SHIP1 was not detected in target tissues of insulin including skeletal muscles and fat cells (8). These results implied the existence of a SHIP1 isozyme responsible for insulin signaling. Accordingly, we and others have cloned a novel 5'-phosphatase named SHIP2 that is predominantly expressed in the target tissues of insulin (9, 10). Although both PI(3,4,5)P3 and PI(3,4)P2 were presumed to relay the insulin signal, overexpression of SHIP2 inhibited insulin-induced glucose uptake and glycogen synthesis in 3T3-L1 adipocytes and L6 myotubes via the 5'-phosphatase activity (11, 12). Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity without affecting biological systems other than insulin signaling (13). These reports indicate that SHIP2 is a physiologically important negative regulator relatively specific for insulin signaling, and that downstream molecules of PI3-kinase are preferentially activated by PI(3,4,5)P3 rather than by PI(3,4)P2. Therefore, elucidation of the mechanisms by which SHIP2 functioning is regulated would be important for further understanding the novel mechanism in the physiological control of insulin signaling. Based on the study with SHIP1, a change of cellular localization, rather than enzymatic activity after ligand stimulation, may be crucial for SHIP2 functioning, probably by enabling access to its target molecules (14).
In the present study, to investigate the importance of the localization of SHIP2 in plasma membrane for its functioning, we constructed an expression plasmid encoding a chimera of c-src myristoylation signal and SHIP2 (myr-SHIP2). Because Akt is one of the downstream molecules of PI3-kinase important for the metabolic action of insulin and Akt is activated by phosphorylation at Ser473 and Thr308 (15, 16, 17, 18, 19), the effect of the expression of membrane-targeted myr-SHIP2 on insulin-induced phosphorylation of Akt was studied in Rat1 fibroblasts expressing insulin receptors. In addition, SHIP1 was originally identified as the Shc-binding molecule, and SHIP1 is known to interact with Shc via the SH2 domain and C-terminal tyrosine phosphorylation sites (5, 20, 21, 22, 23). In line with this, we have found that insulin induces the association of SHIP2 with Shc in Rat1 fibroblasts expressing insulin receptors (10). We reasoned that the insulin-induced SHIP2 interaction with Shc might be important for the appropriate localization and functioning of SHIP2. To this end, we examined the effect of 47-mutant SHIP2 with R47Q change (R/Q-SHIP2) and 987-mutant SHIP2 with Y987F change (Y/F-SHIP2) expressions, which were defective in interaction with Shc, on the phosphorylation of Akt. Furthermore, the effect of SHIP2 expression on the inhibition of insulin-induced Akt phosphorylation was compared between the cells with normal and decreased amounts of Shc.
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RESULTS
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Structures of SHIP2 Constructs
A membrane-targeted chimera of C-terminally FLAG-tagged SHIP2 was generated by fusing the enzyme with the N-terminal region of c-src (myr-SHIP2-FLAG). Because SHIP2 is composed of an SH2 domain at the N terminus, a central 5'-phosphatase catalytic domain and a proline-rich region including a phosphotyrosine binding (PTB) domain-binding consensus at the C terminus, we further generated SHIP2-FLAG and myr-SHIP2-FLAG cDNA containing a 47-R/Q mutation in the SH2 domain,
IP mutation in the 5'-phosphatase catalytic domain, or 987-Y/F mutation in the C terminus to examine the role of each site (Fig. 1
). Each plasmid was transiently transfected into HIRc cells by the lipofection method. The expression of SHIP2 was confirmed by immunoblot analysis with anti-SHIP2 antibody and anti-FLAG antibody.
Identification of SHIP2 Tyr987 as a Phosphorylation Site by Insulin Stimulation
Because the SHIP2 Tyr987 residue is located within the PTB consensus motif, we examined its role in the insulin-induced phosphorylation of SHIP2. Tyrosine phosphorylation of WT- and Y/F-SHIP2 was minimal in the basal state. Insulin induced the apparent tyrosine phosphorylation of WT-SHIP2, whereas tyrosine phosphorylation of Y/F-SHIP2 was not detectable after insulin stimulation (Fig. 2A
). The amounts of WT- and Y/F-SHIP2 were confirmed to be equal by immunoblot analysis with anti-FLAG antibody (Fig. 2B
).

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Figure 2. Identification of Insulin-Induced Tyrosine Phosphorylation Site of SHIP2
HIRc cells transfected with WT-SHIP2 or Y/F-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for 5 min. The cells were solubilized, and the cell lysates were immunoprecipitated with anti-FLAG antibody. The precipitates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibody (A) or anti-FLAG antibody (B). Results are representative of three separate experiments.
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Role of SH2 Domain and C Terminus Tyrosine Phosphorylation Site of SHIP2 in Insulin-Induced Akt Phosphorylation
To investigate the role of the SH2 domain and C terminus tyrosine phosphorylation site of SHIP2, HIRc cells were transiently transfected with an expression plasmid encoding WT-SHIP2, R/Q-SHIP2, or Y/F-SHIP2, or with an empty plasmid. Because Akt is activated by phosphorylation at Ser473 and Thr308, the effect of SHIP2 expression on insulin-induced Akt phosphorylation was examined by immunoblotting with antiphospho-specific Akt antibody (16, 18, 19). Each transfected cell expressed 4-fold the amount of WT-, R/Q-, and Y/F-SHIP2 compared with endogenous SHIP2. Insulin induced the phosphorylation of Akt at both Ser473 and Thr308 in the original HIRc cells. Overexpression of WT-SHIP2 inhibited the insulin-induced phosphorylation at both residues. The expression of R/Q- and Y/F-SHIP2 also inhibited the insulin-induced Akt phosphorylation. However, the degree of inhibition was less than that seen by WT-SHIP2 expression. Equal amounts of protein loaded were confirmed by immunoblotting the cell lysates with anti-Akt antibody (Fig. 3A
). These results are summarized in Fig. 3B
. After 7 min of insulin stimulation, the phosphorylation of Akt at Ser473 and Thr308 was decreased to 46 ± 1% and 29 ± 2% by the expression of WT-SHIP2, to 84 ± 1% and 51 ± 2% by the expression of R/Q-SHIP2, and to 64 ± 2% and 37 ± 3% by the expression of Y/F-SHIP2, respectively, compared with that in the original HIRc cells. It is of note that the partial inhibition of the Akt phosphorylation seen in the transient expression of WT-SHIP2 did not primarily originate from low transfection efficiency, because insulin-induced phosphorylation of Akt was still not completely inhibited in the cells stably overexpressing WT-SHIP2 (data not shown).

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Figure 3. Role of SH2 Domain and C-Terminal Tyrosine Phosphorylation Site of SHIP2 in Insulin-Induced Akt Phosphorylation
A, HIRc cells transfected with an empty vector, WT-SHIP2, R/Q-SHIP2, or Y/F-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for 7 min. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphospho-Ser473 Akt antibody, antiphospho-Thr308 Akt antibody, anti-SHIP2 antibody, anti-FLAG antibody, or anti-Akt antibody. B, The amount of Akt phosphorylated at Ser473 and Thr308 was quantified by densitometry. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. insulin-induced phosphorylation of Akt at the respective residues in control cells.
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Effect of myr-SHIP2 Expression on Insulin-Induced Akt Phosphorylation
The enzymatic function of signaling molecules can be regulated by redistributing their subcellular localization to the plasma membrane, by which enzymes can gain access to the substrate. Thus, SHIP1 has been shown to be recruited to the membrane by the binding of its SH2 domain to the phosphorylated immunoreceptor tyrosine-based inhibitory motif of Fc
-RIIb or cytoplasmic tyrosine residues of several receptors (24, 25, 26). Therefore, to investigate whether the membrane localization of SHIP2 is important for the inhibition of insulin-induced Akt phosphorylation, a membrane-targeted chimera of SHIP2-FLAG was generated by fusing the enzyme with the N-terminal myristoylation signal of c-src (myr-SHIP2-FLAG). HIRc cells were transfected with an expression plasmid encoding WT-SHIP2 or myr-SHIP2, or an empty plasmid. These transfected cells were incubated with 17 nM insulin for specific periods, and the cell lysates were subjected to immunoblotting with antiphospho-Akt antibody. Because the maximal expression of myr-SHIP2 was low and about 10% compared with endogenous SHIP2, we adjusted the expression level of WT-SHIP2 identical with that of myr-SHIP2, which was confirmed by immunoblotting with anti-FLAG antibody. Total SHIP2 amounts detected by anti-SHIP2 antibody were only slightly affected by expression of WT-SHIP2 and myr-SHIP2 under these experimental conditions. Equal protein amounts loaded among the samples were also confirmed by immunoblotting with anti-Akt antibody. Insulin-induced Akt phosphorylation was not apparently affected at this low level of WT-SHIP2 expression. Interestingly, despite the low levels of myr-SHIP2, insulin-induced Akt phosphorylation was efficiently inhibited by the expression (Fig. 4A
). These results are summarized in Fig. 4B
. After 7 min of insulin stimulation, Akt phosphorylation was not affected by transfection with WT-SHIP2, whereas the expression of myr-SHIP2 significantly inhibited insulin-induced Akt phosphorylation at Ser473 and Thr308 to 44 ± 16% and 7 ± 2%, respectively, compared with control HIRc cells.

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Figure 4. Effect of myr-SHIP2 Expression on Insulin-Induced Akt Phosphorylation
A, HIRc cells transfected with an empty vector, WT-SHIP2, or myr-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for the indicated times. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphospho-Ser473 Akt antibody, antiphospho-Thr308 Akt antibody, anti-SHIP2 antibody, anti-FLAG antibody, or anti-Akt antibody. B, The amount of Akt phosphorylated at Ser473 and Thr308 was quantified by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-induced phosphorylation of Akt at the respective residues at the indicated times in control cells.
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Effect of myr-
IP-SHIP2 Expression on Insulin-Induced Akt Phosphorylation
We next examined the role of the 5'-phosphatase activity of membrane-targeted SHIP2 in the insulin-induced phosphorylation of Akt. To this end, the effect of expression of a 5'-phosphatase-defective myr-SHIP2 (myr-
IP-SHIP2) on insulin-induced Akt phosphorylation was examined. Again, expression levels were adjusted to be the same between myr-SHIP2- and myr-
IP-SHIP2-expressing HIRc cells as shown in Fig. 4
. The decrease in insulin-induced Akt phosphorylation seen on expression of myr-SHIP2 was not observed on expression of myr-
IP-SHIP2 (Fig. 5A
). Thus, the level of phosphorylation at both Ser473 and Thr308 was comparable between control HIRc cells and myr-
IP-SHIP2-expressing HIRc cells (Fig. 5B
).

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Figure 5. Effect of myr- IP-SHIP2 Expression on Insulin-Induced Akt Phosphorylation
A, HIRc cells transfected with an empty vector, myr-SHIP2, or myr- IP-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for the indicated times. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphospho-Ser473 Akt antibody, antiphospho-Thr308 Akt antibody, anti-SHIP2 antibody, anti-FLAG antibody, or anti-Akt antibody. B, The amount of Akt phosphorylated at Ser473 and Thr308 was quantified by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-induced phosphorylation of Akt at the respective residues at the indicated times in control cells.
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Effect of Addition of Myristoylation Signal to R/Q-SHIP2 and Y/F-SHIP2 on Insulin-Induced Akt Phosphorylation
To further investigate the role of membrane localization in SHIP2 functioning, we constructed myr-SHIP2 with the 47-R/Q mutation (myr-R/Q-SHIP2) or myr-SHIP2 with the 987-Y/F mutation (myr-Y/F-SHIP2). HIRc cells were transfected with an expression plasmid encoding myr-SHIP2, myr-R/Q-SHIP2, or myr-Y/F-SHIP2, or with empty plasmid. The cells were incubated with 17 nM insulin for 10 min, and the cell lysates were subjected to immunoblotting with antiphospho-Akt antibody as shown in Fig. 6A
. Interestingly, the expression of myr-R/Q-SHIP2 and myr-Y/F-SHIP2 efficiently inhibited insulin-induced Akt phosphorylation at Ser473 and Thr308 to the same level as the expression of myr-SHIP2. After 10 min of insulin stimulation, Akt phosphorylation at Ser473 and Thr308 was decreased to 42 ± 5% and 57 ± 9% in myr-SHIP2 cells, to 34 ± 4% and 56 ± 4% in myr-R/Q-SHIP2 cells, and to 35 ± 2% and 32 ± 18% in myr-Y/F-SHIP2 cells, respectively (Fig. 6B
). Expression levels of myr-SHIP2, myr-R/Q-SHIP2, and myr-Y/F-SHIP2 were confirmed to be similar by immunoblotting the cell lysates with anti-FLAG antibody, and identical amounts of loaded protein were confirmed by immunoblotting with anti-Akt antibody.

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Figure 6. Effect of Myristoylation Signal-Added R/Q-SHIP2 and Y/FSHIP2 Expression on Insulin-Induced Akt Phosphorylation
A, HIRc cells transfected with WT-SHIP2, myr-SHIP2, myr-R/Q-SHIP2, or myr-Y/F-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for 10 min. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphospho-Ser473 Akt antibody, antiphospho-Thr308 Akt antibody, anti-SHIP2 antibody, anti-FLAG antibody, or anti-Akt antibody. B, The amount of Akt phosphorylated at Ser473 and Thr308 was quantified by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-induced phosphorylation of Akt at the respective residues in control cells.
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Role of the SH2 Domain and C Terminus Tyrosine Phosphorylation Site of SHIP2 in Its Association with Shc
SHIP2 appears to possess two potential sites that associate with Shc. One is the phosphorylated Tyr987 residue in the NPXY motif, which interacts with the PTB domain of Shc (27). Based on studies with SHIP1, the other may be the SH2 domain, which interacts with the phosphorylated tyrosine residue in the YXXM motif of Shc (9, 10). To directly investigate the relative importance of these two sites of SHIP2 in the interaction with Shc, the insulin-induced association of Shc with R/Q-SHIP2 and Y/F-SHIP2 was examined. HIRc cells were transiently transfected with WT-SHIP2, R/Q-SHIP2, or Y/F-SHIP2, and then incubated with 17 nM insulin for 5 min. Next, the cell lysates were immunoprecipitated with anti-Shc antibody, and the precipitates were analyzed by immunoblotting with anti-FLAG antibody as shown in Fig. 7A
. In the basal state, SHIP2 association with Shc was barely detected in any of the transfected cells. After insulin stimulation, an apparent association of WT-SHIP2 with Shc was observed. Although R/Q-SHIP2 and Y/F-SHIP2 associated with Shc after the stimulation, the extent of their association was decreased compared with that of WT-SHIP2. In addition, the insulin-induced SHIP2 association with Shc was more decreased in the R/Q mutant than the Y/F mutant. These results are summarized in Fig. 7B
. The association of SHIP2 with Shc was decreased to 49 ± 3% by R/Q-SHIP2 expression and to 78 ± 2% by Y/F-SHIP2 expression compared with that seen on expression of WT-SHIP2. Similar levels of expression of these SHIP2 constructs were confirmed by immunoblotting the cell lysates with anti-FLAG antibody, and equal amounts of loaded protein were confirmed by immunoblotting with anti-Shc antibody (Fig. 7A
).

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Figure 7. Role of SH2 Domain and C-Terminal Tyrosine Phosphorylation Site of SHIP2 in Insulin-Induced SHIP2 Association with Shc
A, HIRc cells transfected with WT-SHIP2, R/Q-SHIP2, or Y/F-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for 5 min. The cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates or total cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibody or anti-Shc antibody. B, The amount of SHIP2 associated with Shc was quantified by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-induced SHIP2-Shc association in WT-Shc cells.
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Effect of Shc Amount on Insulin-Induced SHIP2 Association with Shc
We further examined whether the amount of Shc affects the insulin-induced association of SHIP2 with Shc. To this end, we employed HIRc cells overexpressing WT-Shc (WT-Shc) and expressing antisense Shc mRNA (AS-Shc). The amount of Shc expressed in AS-Shc cells was decreased to 50%, whereas 5 times more WT-Shc was expressed in WT-Shc cells than the original HIRc cells. SHIP2 was minimally associated with Shc in the basal state in all cell lines. The insulin-induced association of SHIP2 with Shc was decreased to 32 ± 3% in AS-Shc cells, whereas it was increased to 140 ± 3% in WT-Shc cells compared with the original HIRc cells (Fig. 8
, A and B). The amounts of SHIP2 in the transfected cells were confirmed to be identical by immunoblotting with anti-SHIP2 antibody.

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Figure 8. Effect of AS-Shc and WT-Shc Expression on Insulin-Induced SHIP2 Association with Shc
A, HIRc, AS-Shc, and WT-Shc cells were serum starved for 16 h and then incubated with 17 nM insulin for 5 min. The cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates or total cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-SHIP2 antibody or anti-Shc antibody. B, The amount of SHIP2 associated with Shc was quantified by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-induced SHIP2-Shc association in HIRc cells.
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Effect of Shc Amounts on the Inhibition of Insulin-Induced Akt Phosphorylation by SHIP2
Finally, we compared the effect of WT-SHIP2 overexpression on insulin-induced Akt phosphorylation in the original HIRc cells and AS-Shc cells. The phosphorylation at Ser473 and Thr308 was not significantly different between HIRc and AS-Shc cells. A 5-fold excess of WT-SHIP2 efficiently inhibited the insulin-induced phosphorylation of Akt in HIRc cells. In contrast, the degree of inhibition by the overexpression of WT-SHIP2 was decreased by a 50% reduction in the amount of endogenous Shc in AS-Shc cells (Fig. 9A
). Thus, overexpression of WT-SHIP2 decreased the insulin-induced Akt phosphorylation at Ser473 and Thr308 to 12 ± 1% and 21 ± 3%, respectively, in HIRc cells, compared with only 70 ± 4% and 61 ± 6%, respectively, in AS-Shc cells (Fig. 9A
). Because the extent of overexpression of WT-SHIP2 appeared to be relatively higher, the degree of inhibition of the Akt phosphorylation in HIRc cells might be greater in Fig. 9
compared with that in Fig. 3
.

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Figure 9. Effect of WT-SHIP2 Overexpression on Insulin-Induced Akt Phosphorylation in HIRc and AS-Shc Cells
A, HIRc and AS-Shc cells transfected with WT-SHIP2 were serum starved for 16 h and then incubated with 17 nM insulin for 5 min. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphospho-Ser473 Akt antibody, antiphospho-Thr308 Akt antibody, anti-SHIP2 antibody, anti-FLAG antibody, anti-Shc antibody, or anti-Akt antibody. B, The amount of Akt phosphorylated at Ser473 and Thr308 was quantified by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. insulin-induced phosphorylation of Akt at the respective residues in AS-Shc cells without WT-SHIP2 overexpression.
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DISCUSSION
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PI(3,4,5)P3 produced by the activated PI3-kinase is known to be an important second messenger to signal downstream molecules for metabolic action of insulin (28, 29). Recently, it has been shown that the PI(3,4,5)P3 produced in response to insulin can be hydrolyzed either to PI(4,5)P2 by a 3'-phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), or to PI(3,4)P2 by a 5'-phosphatase SHIP2 (11, 30). Facilitation of the hydrolysis of PI(3,4,5)P3 to PI(4,5)P2 by overexpression of PTEN inhibited the insulin-induced Akt activation and glucose uptake in 3T3-L1 adipocytes (31). However, the insulin-induced glucose uptake was not affected by the expression of a catalytically inactive PTEN, which functions in a dominant-negative manner (32). Taken together, PTEN does not appear to be a physiologically important negative regulator of insulin signaling. Facilitation of the hydrolysis of PI(3,4,5)P3 to PI(3,4)P2 by SHIP2 overexpression also inhibited insulin-induced Akt activity resulting in decreased Glut4 translocation and glucose uptake in 3T3-L1 adipocytes and L6- myotubes (11, 12). In addition to Akt, downstream molecules of PI3-kinase including protein kinase C-
and protein phosphatase 1, and phosphorylation of glycogen synthase kinase-3ß, were all negatively regulated by WT-SHIP2 (11). Importantly, the insulin-induced activation of Akt, protein kinase C-
, and protein phosphatase 1, and the phosphorylation of glycogen synthase kinase-3ß, were enhanced by the expression of a dominant-negative mutant of SHIP2 (
IP-SHIP2) (11). Furthermore, it has been shown that targeted disruption of mice with SHIP2 increases insulin sensitivity (13). Based on the histological analysis of mice, biological systems other than insulin signaling did not appear to be affected, indicating a relatively specific involvement of SHIP2 in insulin signaling (13). Taken together, previous reports clearly indicate the importance of SHIP2 as a novel physiological negative regulator of insulin signaling (11, 12, 13). Therefore, clarification of the molecular mechanisms by which SHIP2 functions to regulate downstream molecules of PI3-kinase would be of particular importance for further understanding novel physiological systems in the control of insulin signaling. Because Akt is one of the downstream molecules of PI3-kinase important for the biological action of insulin (15, 17) and Akt is primarily activated by phosphorylation at Thr308 and Ser473 (16, 18, 19), we clarified how SHIP2 regulates the insulin-induced phosphorylation of Akt.
It is reported that SHIP1, via the SH2 domain, binds to the phosphorylated Tyr317 residue of Shc, and SHIP1, via the C terminus tyrosine phosphorylation sites, binds to the PTB domain of Shc (22, 23). Accordingly, SHIP1 possesses dual binding sites to interact with Shc. Because of the structural similarities between SHIP1 and SHIP2, one can speculate that SHIP2 interacts with Shc in a similar manner. Concerning the tyrosine phosphorylation site of SHIP2, we identified Tyr987 in the NPXY motif as a critical phosphorylation site of SHIP2, because the mutant SHIP2 with a 987-Y/F substitution was not tyrosine phosphorylated after insulin stimulation (Fig. 2
). In contrast, it was reported that the truncated form of SHIP2 lacking the NPAY site was still tyrosine phosphorylated after EGF stimulation (33), indicating the existence of alternative tyrosine phosphorylation site(s). The reason for this difference is unknown. However, it may arise from different growth factors and/or cells used for the study. In addition, we cannot rule out the possibility that tyrosine residue(s) other than Tyr987 is phosphorylated, albeit to a lesser extent, although it is not detected in our experimental system.
Our results showed that insulin treatment induced SHIP2 association with Shc dependent on the amount of Shc, and that the content of insulin-induced R/Q-SHIP2 and Y/F-SHIP2 associated with Shc was decreased compared with that of WT-SHIP2. These results indicate the importance of the SH2 domain and C-terminal tyrosine phosphorylation site of SHIP2 for interaction with Shc. SHIP2 interaction with Shc via its SH2 domain appears to be more important than that via its C-terminal tyrosine residue. The importance of the SH2 domain of SHIP2 was further indicated by the fact that the level of insulin-induced tyrosine phosphorylation of R/Q-SHIP2 was less than that of WT-SHIP2 (data not shown). It is possible that the SH2 domain of SHIP2 indirectly affects the SHIP2-Shc interaction via Tyr987. In addition, antiphospho-specific SHIP2 antibody poorly recognized a mutant SHIP2 deleted of the SH2 domain after EGF stimulation (33). In contrast, the phosphorylated peptide comprising the NPXY motif of SHIP2 was efficiently able to bind to Shc in CHO-IR cells, and the SH2 domain of SHIP2 had weak interaction with Shc after stimulation with EGF in Cos-7 cells (33). Although the relative importance of the SH2 domain vs. the C-terminal tyrosine residue for interaction with Shc remains to be elucidated, the degree of the inhibition of insulin-induced phosphorylation of Akt was decreased by R/Q-SHIP2 and Y/F-SHIP2 compared with WT-SHIP2. In addition, the extent of the inhibition was correlated with the degree to which SHIP2 associated with Shc. These results indicate that the association of SHIP2 with Shc is a key to triggering an efficient inhibitory effect of SHIP2 on insulin-induced phosphorylation of Akt. This notion was further reinforced by the fact that the effect of WT-SHIP2 overexpression on the inhibition of insulin-induced Akt phosphorylation was decreased in AS-Shc mRNA-expressing cells compared with original HIRc cells.
The reduction of SHIP2 association with Shc in AS-Shc did not significantly affect insulin-induced phosphorylation of Akt unless WT-SHIP2 was overexpressed as can be seen in Fig. 9
. Theoretically, the reduction of the amount of Shc and SHIP2-Shc association leads to the increased phosphorylation of Akt by the reduction of SHIP2 functioning. However, we assume that the phosphorylation of Akt was induced close to the maximal level upon insulin stimulation. Further enhancement of the already maximally stimulated Akt phosphorylation by the reduction of SHIP2 function may be difficult. In contrast, the restoration of the decreased phosphorylation of Akt by WT-SHIP2 expression appears to be readily seen by the reduction of SHIP2 function in AS-Shc cells.
The relocalization of SHIP1 in the vicinity of the plasma membrane appears to be critical for its functioning in hematopoietic cells (34, 5). Given this, we reasoned that SHIP2 elicits its function via similar mechanisms. Therefore, we addressed the importance of the plasma membrane localization of SHIP2 in the regulation of insulin-induced Akt phosphorylation by expressing myr-SHIP2. Unexpectedly, the expression level of SHIP2 constructs with the myristoylation signal obtained using our conventional transient transfection method was much lower than that of various SHIP2 constructs without the signal. Although the reason for this is unclear, the low expression level of myr-SHIP2 appears to be due to the characteristics of myr-SHIP2 itself rather than any technical problem, because the appropriate construction of cDNAs was confirmed by sequencing. Along this line, the SHIP family of proteins is also known to play a negative regulatory role in cell growth and development (10, 23, 35, 36, 37, 38). Therefore, a possible excess of functional SHIP2 protein would unfavorably affect cell survival in addition to the control of insulins metabolic signal. It is of note that the level of myr-SHIP2 expression did not affect cell viability, because myr-SHIP2 did not interfere with the early events of insulin signaling until PI3-kinase activation (data not shown), which was consistent with our previous experiments on WT-SHIP2 expression (10). Regardless of the mechanisms, this low expression level of myr-SHIP2 is enough to efficiently inhibit insulin-induced Akt phosphorylation to the level obtained with a much higher expression level of WT-SHIP2. Thus, localization of SHIP2 at the plasma membrane, albeit to a lesser extent of the expression, appears to be required to efficiently inhibit insulin-induced Akt phosphorylation. In addition, our results showed that the phosphatase-defective myr-SHIP2 did not function even though it is localized at the plasma membrane. Therefore, we conclude that membrane localization of SHIP2 is important for its functioning via 5'-phosphatase activity to regulate insulin-induced Akt phosphorylation. The expressed phosphatase-defective myr-SHIP2 did not act with the dominant-negative manner, at least in the Akt phosphorylation, of the present study using Rat1 fibroblasts overexpressing insulin receptors, whereas expression of the catalytically defective SHIP2 functioned in a dominant-negative fashion in 3T3-L1 adipocytes (11). These differences may be due to the cell type specificity or to the difference in membrane localization by addition of the myristoylation signal. Furthermore, myr-R/Q-SHIP2 and myr-Y/F-SHIP2 also efficiently inhibited insulin-induced Akt phosphorylation similar to myr-SHIP2 and much larger amounts of WT-SHIP2. These results indicate that the SH2 domain and the C-terminal tyrosine phosphorylation site are required for the membrane localization, at least in part, by interaction with Shc, and that these two sites do not directly affect the 5'-phosphatase activity of SHIP2. If our results with expression studies of various SHIP2 constructs can apply to the endogenous SHIP2, it may be possible to propose a scenario for SHIP2 functioning, in which insulin stimulation leads to the relocalization of SHIP2 from cytosol to plasma membrane via interaction with Shc; in this way, SHIP2 can gain access to the substrate PI(3,4,5)P3 resulting in its hydrolysis to PI(3,4)P2, which in turn regulates Akt activity in the control of insulin signaling. This hypothesis is supported by a recent report showing that the ability of SHIP2 mutants bearing the SH2 domain or C-terminal NPAY motif to hydrolyze PI(3,4,5)P3 to PI(3,4)P2 was similar to that of WT-SHIP2 when these mutants were directly incubated with PI(3,4,5)P3 in vitro. However, the ability of SHIP2 mutants in the expressed cells was low compared with that of WT-SHIP2 (38).
In summary, these results indicate that the membrane localization of SHIP2 with its 5'-phosphatase activity is required for negative regulation of insulin-induced Akt phosphorylation, and that the localization is regulated, at least in part, by the association of SHIP2 with Shc in Rat1 fibroblasts. Because SHIP2 appears to be a physiological negative regulator relatively specific for insulin signaling, clarification of the mechanisms by which SHIP2 functions would be of a particular worth for the development of new therapeutic drugs to improve the insulin resistance seen in type 2 diabetes.
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MATERIALS AND METHODS
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Materials
Human crystal insulin was provided by Novo Nordisk Pharma Ltd. (Copenhagen, Denmark). The two polyclonal anti-SHIP2 antibodies were described previously (10). A monoclonal anti-FLAG antibody (M2) was purchased from Sigma (St. Louis, MO). A monoclonal anti-Shc antibody and a polyclonal anti-Shc antibody were obtained from Transduction Laboratories, Inc. (Lexington, KY). A polyclonal anti-Thr308 phospho-specific Akt antibody and a polyclonal anti-Ser473 phospho-specific Akt antibody were purchased from New England Biolabs, Inc. (Beverly, MA). A polyclonal anti-Akt antibody and a monoclonal antiphosphotyrosine antibody (PY99) were bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other reagents were of analytical grade and purchased from Sigma or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Plasmid Construction
We performed PCR with a 5'-primer containing a HindIII restriction site (underlined), 5'-AAGCTTATGGGGAGTAGCAAGAGC-3', and a 3'-primer containing a SalI restriction site (underlined), 5'-GTCGACCGGCGCTGGCTGGGGTC-3', to isolate the myristoylation signal MGSSKSKPKDPSQRR from c-src. The DNA fragment generated was inserted into the HindIII/SalI site of the mammalian expression vector pFLAG-CMV-5b (Sigma) to yield pMyr-FLAG. A SHIP2 fragment that has neither the initiation methionine codon nor the termination codon was prepared with a 5'-primer containing a SalI restriction site (underlined), 5'-GGGTCGACGACTAGTGCCTCAGTGTGTGGGGCACCGAGTCCCGGG-3', and a 3'-primer containing a SalI restriction site (underlined), 5'-CGTCGACCCGGTACCCTCCTCTGCCAAGTCTTCTTGGATGCTCTC-3', and subcloned into the SalI site of the pMyr-FLAG expression vector to allow in-frame fusion to the SHIP2 sequence (pMyr-SHIP2-FLAG). A WT-SHIP2-fused FLAG expression vector (pSHIP2-FLAG) was constructed as described previously (10). The SH2 domain 47-R/Q mutant (R/Q-SHIP2), PTB domain binding motif 987-Y/F mutant (Y/F-SHIP2), and 5'-phosphatase-defective mutant (
IP-SHIP2) were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with 5'-GCGATGGAAGCTTCCTGGTGCAAGACAGC-3' (sense) and 5'-GCTGTCTTGCACCAGGAAGCTTCCATCGC-3' (antisense), 5'-AATAACCCTGCCTTCTACGTACTTGAAGGG-3' (sense) and 5'-CCCTTCAAGTACGTAGAAGGCAGGGTTATT-3' (antisense), and 5'-ACCAATGTGGCTTCATGGTGTGCCGGAATTCTATGG-3' (sense) and 5'-CCATAGAATTCCGGCACACCATGAAGCCACATTGGT-3' (antisense), respectively. The presence of appropriate mutations in these constructs was confirmed by sequencing.
Cell Culture and DNA Transfection
Rat1 fibroblasts expressing 1 x 106 human insulin receptors per cell (HIRc) were kindly supplied by Dr. J. M. Olefsky (University of California, San Diego, CA) and were maintained in DMEM/F-12 medium supplemented with 10% fetal calf serum (39). Various SHIP2 constructs were transiently expressed utilizing the TransIT-LT1 transfection reagent according to the manufacturers instructions (Pan Vera Co., Madison, WI). The transfection efficiency of these SHIP2 constructs was the same, and about 40% by detection with immunofluorescent staining using anti-FLAG antibody. HIRc cells stably overexpressing WT-Shc and expressing AS-Shc mRNA were described previously (40).
Immunoprecipitation and Western Blotting
The transfected cells grown in 6-cm dishes were serum starved for 16 h. The cells were treated with 17 nM insulin at 37 C for various 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, 1 mM Na3VO4, 160 mM sodium fluoride, 10 µg of aprotinin per ml, 10 µM leupeptin (pH 7.4) for 15 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. The supernatants were immunoprecipitated with various antibodies for 2 h at 4 C. The precipitates or total cell lysates were then separated by 7.5% SDS-PAGE and electrically transferred onto polyvinylidine difluoride membranes. After incubation with the specified antibody, enhanced chemiluminescence detection was performed according to the manufacturers instructions (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Statistical Analysis
The data are represented as means ± SE. P values were determined by Scheffés multiple comparison test, and P < 0.05 was considered statistically significant.
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ACKNOWLEDGMENTS
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FOOTNOTES
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This work was supported in part by the Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (to T.S.), and by Japan Diabetes Foundation (to T.S.).
Abbreviations: AS-Shc, Antisense Shc; myr-SHIP2, chimera of c-src myristoylation signal and SHIP2; myr-
IP-SHIP2, 5'-phosphatase-defective myr-SHIP2; PI3-kinase, phosphatidyl inositol 3-kinase; PI(3,4)P2, phosphatidylinositol-3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol-3,4,5,-triphosphate; PI(4)P, phosphatidylinositol-4-phosphate; PI(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PTB, phosphotyrosine binding; PTEN, phosphatase and tensin homolog deleted on chromosome 10; R/Q-SHIP2, mutant SHIP2 with R47Q change; SH2, Src homology 2; SHIP1 and SHIP2, SH2-containing inositol 5'-phosphatase 1 and 2; WT, wild-type; YIF-SHIP2, mutant SHIP2 with Y987F change.
Received for publication February 25, 2002.
Accepted for publication June 11, 2002.
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