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Tyrosine Phosphorylation-Dependent and -Independent Role of Shc in the Regulation of IGF-1-Induced Mitogenesis and Glycogen Synthesis

First Department of Medicine (T.S., M.I., T.W., H.Ho., H.Hi., T.H., H.I., M.K.) and Department of Clinical Pharmacology (T.S.), Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, 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 functional role of Shc tyrosine phosphorylation in IGF-1 signaling, wild-type (WT)-Shc and Y239,240,317F (3F)-Shc were transiently transfected into L6 myoblasts. IGF-1 signaling was compared among the transfected cells. IGF-1-induced tyrosine phosphorylation of Shc and its subsequent association with Grb2 were increased in WT-Shc cells, whereas they were decreased in 3F-Shc cells compared with those in parental L6 cells. Consistent with their changes, IGF-1-induced MAPK activation and thymidine incorporation were enhanced in WT-Shc cells, whereas they were again decreased in 3F-Shc cells. It is possible that Shc and insulin receptor substrate (IRS)-1 can interact competitively, via their phosphotyrosine binding (PTB) domains, with the activated IGF-1 receptor. In this regard, IGF-1-induced tyrosine phosphorylation of IRS-1 was decreased by overexpressing both WT-Shc and 3F-Shc cells. Consistent with the decrease, IGF-1-induced IRS-1 association with the p85 subunit of PI3K and activation of PI3K and Akt were reduced in both WT-Shc and 3F-Shc cells. As a result, IGF-1-induced glycogen synthesis was also decreased in both cells. Furthermore, expression of Shc PTB domain alone inhibited IGF-1 stimulation of Akt and glycogen synthesis. These results indicate that tyrosine phosphorylation of Shc is important for IGF-1 stimulation of MAPK leading to mitogenesis and that Shc, via its PTB domain, negatively regulates IGF-1-induced glycogen synthesis by competing with IRS-1, which is not relevant to Shc tyrosine phosphorylation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE ACTIVATED IGF-1 receptor phosphorylates various cellular substrates on tyrosine residues (1, 2, 3, 4, 5, 6, 7). One of the substrates of the IGF-1 receptor is Shc (3, 4, 5, 6, 7). Previous studies demonstrated an important role of Shc in IGF-1-induced mitogenesis (7, 8). Shc is composed of three distinct domains containing an amino-terminal region called the phosphotyrosine binding (PTB) domain, a collagen homology (CH) domain, and a carboxyl-terminal SH2 domain (9). Shc has been shown to be important primarily in the process of the activation of p21^ras-MAPK, which plays a pivotal role in mitogenic signal transduction initiated by receptor tyrosine kinases, including the IGF-1 receptor (3, 7, 8, 10). Upon IGF-1 stimulation, tyrosine-phosphorylated Shc associates with Grb2, which forms a complex with Sos, a p21^ras guanine nucleotide exchange factor (8). Shc·Grb2 binding is mediated by the SH2 domain of Grb2 binding to phosphorylated tyrosine residues within the CH domain of Shc (7, 11). Tyr-239, Tyr-240, and Tyr-317 residues of Shc are the possible candidates of responsible phosphorylation sites for Grb2 binding (12). However, it has not been known whether these tyrosine phosphorylations are in fact critical in the mitogenic signaling of IGF-1 in the skeletal muscle.

Insulin receptor substrate (IRS) family of proteins is also tyrosine-phosphorylated after IGF-1 stimulation (1, 2, 3, 4, 5, 6, 7, 8, 13, 14). IRS-1 is the most well characterized among members of the IRS family, and the tyrosine-phosphorylated IRS-1 binds to the SH2 domain of p85 regulatory subunit of PI3K (15, 16, 17). The activated PI3K generates the production of phosphoinositides on the D3 position, which functions as a key mediator in the activation of downstream molecules of PI3K to exert various metabolic effects of IGF-1 (2, 18). Akt is one of such downstream targets (19, 20, 21, 22) and is thought to be a key molecule in IGF-1-induced glycogen synthesis (23, 24). We have previously shown that Shc and IRS-1 are competitive substrates to interact with the insulin receptor (8, 25, 26). This may also occur in the IGF-1 receptor signaling, because insulin and IGF-1 receptors use similar systems to relay the signal intracellularly (1, 2, 3, 4, 8). If this is the case, overexpression of Shc may affect IGF-1-induced glycogen synthesis by modulating the PI3K-dependent pathway leading to glycogen synthesis (23, 27, 28).

In the present study, to clarify the specific role of the tyrosine phosphorylation of Shc in IGF-1 signaling, wild-type (WT)-Shc and Y239,240,317F (3F)-Shc were transiently transfected into L6 myoblasts. Intracellular IGF-1 signaling leading to mitogenesis was compared among the transfected cells. In addition, we examined the impact of Shc overexpression on IGF-1-induced glycogen synthesis. Toward this goal, we further used Shc PTB domain, because Shc and IRS-1 may interact competitively, via their PTB domains, with the IGF-1 receptor.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Human recombinant IGF-1 was the kind gift of Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan). [-³²P]ATP (3000 TBq/mmol) and [³H]thymidine (83 Ci/mmol) were purchased from DuPont NEN (Boston, MA). A polyclonal anti-Shc antibody, a monoclonal anti-p85 antibody, a monoclonal anti-Akt antibody, and a monoclonal antiphosphotyrosine antibody (pY20) were obtained from Transduction Laboratories (Lexington, KY). A polyclonal anti-GST antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A polyclonal antiphospho-specific MAPK antibody was obtained from New England Biolabs, Inc. (Beverly, MA). Enhanced chemiluminescence reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Electrophoresis reagents were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were analytical grade and were purchased from Sigma (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Cell culture
L6 skeletal muscle cells were obtained from American Type Culture Collection (Manassas, VA) and were maintained in -MEM supplemented with 10% FCS.

Plasmid
DNA encoding GST-tagged WT-Shc was generated and subcloned into the pEBG vector as described previously (29, 30, 31). GST-tagged 3F-Shc and PTB domain of Shc were generated by PCR-based mutagenesis, and the mutant Shc DNAs were also subcloned into the pEBG vector. All constructs were sequenced, and the presence of appropriate mutations was confirmed (29, 30, 31).

DNA transfection
Transient transfection into L6 myoblasts was performed using the Effectene transfection reagent (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions . In brief, the cells were washed with sterile PBS, followed by addition of 1.6 ml DMEM to each 35-mm well. Then, preformed complexes (0.8 µg of the indicated DNA in the Effectene reagents per well) were added to each well, and dishes were placed at 37 C in 5% CO₂ (29). Approximately 48 h post transfection, the cells expressing equivalent levels of various Shc proteins were used for further studies. The transfection efficiency of the Shc constructs was about 40% by the immunofluorescent staining with anti-GST antibody, and the efficiency was similar among the expression of various Shc constructs.

Immunoprecipitation and Western blotting
Cells were serum-starved for 24 h and then incubated with 14 nM IGF-1 for various times. The cells were lysed in a solubilizing buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na₃VO₄, 160 mM NaF, pH 7.4, for 15 min at 4 C. Lysates obtained from the same number of cells for each cell line were centrifuged to remove insoluble material, and the supernatants were used for immunoprecipitation with various antibodies for 3 h at 4 C. The immunoprecipitates or whole-cell lysates were separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes by electroblotting. The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA, pH 7.5, for 2 h at 20 C. The membranes were then probed with the specified antibodies for 2 h at 20 C (7). After the membranes were washed 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 according to the manufacturer’s (Amersham Pharmacia Biotech) instructions. To ensure an equal amount of protein loaded for the study, the cell lysates were immunoblotted with anti-Shc antibody. In addition, comparable expression of Shc constructs among the transfected cells was also confirmed by immunoblotting of the cell lysates with anti-GST antibody.

Measurement of MAPK activity
Cell monolayers were starved for 24 h in serum-free DMEM. The cells were treated then with 14 nM IGF-1 for various times at 37 C. The cells were lysed and homogenized in a buffer containing 10 mM Tris, 150 mM NaCl, 2 mM EGTA, 2 mM DTT, 1 mM Na₃VO₄, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, pH 7.4. The cell lysates were centrifuged to remove insoluble materials. The supernatants were used for the measurement of MAPK activity using the Biotrak p42/p44 MAPK enzyme assay system (3). [³²P]ATP incorporation into epidermal growth factor (EGF) receptor-derived peptide for 30 min at 30 C was measured according to the manufacturer’s (Amersham Pharmacia Biotech) instructions.

Measurement of PI3K activity
Serum-starved cells were stimulated with 14 nM IGF-1 at 37 C for various times. The cells were solubilized in a buffer containing 20 mM Tris, 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 100 µM Na₃VO₄, 1% Nonidet-P40, 10% glycerol, 2 mM PMSF, and 10 µg/ml aprotinin, pH 7.6. The cells were centrifuged to remove insoluble materials. The supernatants were immunoprecipitated with anti-IRS-1 antibody for 2 h at 4 C. The precipitates were washed twice with buffer A [Tris-buffered saline, pH 7.6, 1% Nonidet-P40, 100 µM Na₃VO₄, and 1 mM dithiothreitol (DTT)], twice with buffer B (100 mM Tris, pH 7.6, 500 mM LiCl, 100 µM Na₃VO₄, and 1 mM DTT), and twice with buffer C (10 mM Tris, pH 7.6, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT). The phosphorylation reaction was started by adding 20 µl phosphatidylinositol solution containing 0.5 mg/ml phosphatidylinositol, 50 mM HEPES, 1 mM NaH₂PO₄, 1 mM EGTA, pH 7.6, at 20 C, followed by addition of 10 µl of the reaction mixture containing 250 µM [-³²P]ATP (0.37 Mbq/tube), 100 mM HEPES, 50 mM MgCl₂, pH 7.6, for 5 min. The reaction was stopped by the addition of 15 µl 8 M HCl. The products were extracted by adding 130 mM chloroform/methanol (1:1), followed by centrifugation. The organic phase was removed and spotted on Silica gel 60 plates (Merck KGaA, Darmstadt, Germany). The plates were developed and dried (32). The phosphorylated inositol was visualized by autoradiography and quantitated by the Bio-Image Analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Measurement of Akt activity
Cells grown on 10-cm dishes were serum-starved for 16 h in DMEM, washed three times in phosphate-free DMEM, and incubated further for 1 h in the medium. Then, the cells were washed twice with phosphate-free DMEM and incubated with carrier-free [³²P]orthophosphate (1 mCi/ml). The cells were stimulated with various concentrations of IGF-1 for the indicated times at 37 C. The medium was aspirated; the cells were washed twice with ice-cold DMEM buffer and then lysed with 1 ml ice-cold buffer A containing 50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1% vol/vol Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 µM microcystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mM PMSF, 10 µg/ml leupeptin, 0.1% vol/vol 2-mercaptoethanol, pH 7.5. The lysates were centrifuged at 4 C for 10 min at 13,000 x g, and the supernatants were incubated for 30 min on a shaking platform with 50 µl protein G-Sepharose coupled to 50 µg preimmune sheep IgG. The samples were centrifuged for 2 min at 13,000 x g, and the supernatants were incubated further for 60 min with 30 µl protein G-Sepharose covalently coupled to 60 µg Akt antibody. The protein G-Sepharose Akt antibody complex was washed three times with buffer A containing 0.5 M NaCl and twice with 50 mM Tris-HCl, 0.1 mM EGTA, 0.1% vol/vol 2-mercaptoethanol, pH 7.5. Kinase assay was performed on the immune pellets by addition of 50 mM Tris, 10 mM MgCl₂, 1 mM DTT, 5 µM ATP (2 µCi), 30 µM Crosstide, pH 7.5. After 30 min at 22 C, samples were absorbed on phosphocellulose p81 paper and extensively washed in 1% orthophosphoric acid solution. Radioactivity associated to the paper was counted (33).

Thymidine incorporation
Cells were grown to confluence in 24 multiwell culture plates and serum-starved for 24 h. After stimulation of the cells with various concentrations of IGF-1 for 20 h, 1 µCi of [³H]thymidine was added for 4 h. The cells were washed twice with ice-cold PBS, twice with ice-cold 10% trichloroacetic acid, and once with 95% ethanol. The cells were dissolved in 1 N NaOH, neutralized with 1 N HCl, and counted in a liquid scintillation counter (26). In thymidine incorporation studies, comparable expression of Shc constructs was ensured by immunoblotting of the cell lysates that were obtained from a separate set of the transfected cells with anti-GST antibody.

Glucose incorporation into glycogen
Confluent cell monolayers were incubated in glucose-free DMEM for 3 h. Cells were stimulated by IGF-1 for 2 h at 37 C with 5 mM glucose and D-[¹⁴C]glucose. The cell monolayers were washed three times with PBS and solubilized with 30% KOH solution for 30 min at 37 C. After the sample was boiled for 30 min with carrier glycogen, glycogen was precipitated by the addition of ethanol, and radioactivity of the precipitates was counted (28). In glucose incorporation into glycogen studies, comparable expression of Shc constructs was also confirmed by immunoblotting of the cell lysates obtained from a separate set of the transfected cells with anti-GST antibody.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of exogenous WT and mutant Shc in L6 myoblasts
To examine the role of Shc in IGF-1 signaling, we transiently transfected pEBG (empty vector), WT-Shc, and 3F-Shc into L6 myoblasts (Fig. 1A). Each transfected cell expressed a 4-fold greater amount of WT-Shc and 3F-Shc compared with that of endogenous Shc in L6 myoblasts. In addition, the same amount of endogenous Shc among original L6, WT-Shc, and 3F-Shc cells was shown to demonstrate that equivalent amounts of protein were loaded (Fig. 1B).

View larger version (45K): [in this window] [in a new window]   Figure 1. Construction of expression plasmids and expression of exogenous WT and mutant Shc. A, The Shc cDNA and the mutant Shc encoding Tyr-239,240,317-Phe mutation were subcloned into pEBG. Schematic structures of Shc are shown. The three domains of Shc are a PTB domain, a CH domain containing the tyrosine phosphorylation sites, and a carboxyl-terminal SH2 domain, as indicated. B, Mock transfected L6 myoblasts, WT-Shc cells, and 3F-Shc cells were solubilized, and expression of GST-tagged exogenous Shc and endogenous Shc in the cell lysates was analyzed by immunoblotting with anti-Shc antibody. Results are representative of three separate experiments.

View larger version (28K): [in this window] [in a new window]   Figure 2. IGF-1-induced tyrosine phosphorylation of Shc in the transfected cells. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h and then treated with 14 nM IGF-1 for 5 min. The cells were solubilized, and the cell lysates were immunoprecipitated with anti-Shc antibody. Then, the immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibody. A, Representative results are shown. B, The level of tyrosine-phosphorylated Shc was quantitated by densitometry. Results are the mean ± SE of five separate experiments.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effects of Shc overexpression on IGF-1-induced Shc association with Grb2. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h and then treated with 14 nM IGF-1 for 5 min. The cell lysates were immunoprecipitated with anti-Shc antibody. The immunoprecipitates were then subjected to SDS-PAGE and analyzed by immunoblotting with anti-Grb2 antibody. A, Representative results are shown. Molecular mass of Grb2 (25 kDa) is shown by an arrow. B, The amount of Grb2 associated with Shc was quantitated by densitometry. Results are the mean ± SE of six separate experiments.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of Shc overexpression on IGF-1-induced MAPK activation. A, L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h and then treated with 14 nM IGF-1 for 5 min. The cell lysates were immunoblotted with antiphospho-specific MAPK antibody. Representative results are shown. Molecular mass of p42 MAPK (42 kDa) is shown by an arrow. B, The amount of phosphorylated p42 MAPK was analyzed with densitometry. Results are the mean ± SE of three separate experiments. C, Time course of MAPK activity toward EGF receptor-derived peptide as a substrate was measured as described in Materials and Methods. Results are the mean ± SE of three separate experiments.

View larger version (25K): [in this window] [in a new window]   Figure 5. Effects of Shc overexpression on IGF-1-induced thymidine incorporation. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h. Thymidine incorporation in the transfected cells was assayed as described under Materials and Methods. Dose-response curves for IGF-1 stimulation of thymidine incorporation are shown. Results are the mean ± SE of three separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: mock, b =5191 ± 384 dpm/1 x 106 cells, and m = 4.23 ± 0.28-fold; WT-Shc, b = 4953 ± 172 dpm/1 x 106 cells, and m = 5.41 ± 0.24-fold; 3F-Shc, b = 4715 ± 238 dpm/1 x 106 cells, and m = 3.51 ± 0.22-fold.

View larger version (39K): [in this window] [in a new window]   Figure 6. Effects of Shc overexpression on tyrosine phosphorylation of IRS-1. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h and then treated with 14 nM IGF-1 for the indicated times. The cells were solubilized, and the cell lysates were subjected to SDS-PAGE and were analyzed by immunoblotting with anti-phosphotyrosine antibody. A, Representative results are shown. Molecular mass of IRS-1 (180 kDa) is shown by an arrow. B, IRS-1 phosphorylation was quantitated by densitometry. Results are the mean ± SE of three separate experiments.

View larger version (41K): [in this window] [in a new window]   Figure 7. Effects of Shc overexpression on IRS-1 association with the p85 regulatory subunit of PI3K. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h and then treated with 14 nM IGF-1 for 5 min. The cell lysates were immunoprecipitated with IRS-1 antibody. Then, the immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-p85 antibody. A, Representative results are shown. Molecular mass of the p85 (85 kDa) subunit is shown by an arrow. B, The amount of the p85 subunit associated with IRS-1 was quantitated by densitometry. Results are the mean ± SE of four separate experiments.

View larger version (14K): [in this window] [in a new window]   Figure 8. Effects of Shc overexpression on PI3K activity. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h and then treated with 14 nM IGF-1 for 5 min. The cell lysates were immunoprecipitated with IRS-1 antibody. PI3K activity in the anti-IRS-1 immunoprecipitates was assayed as described under Materials and Methods. A, Representative results are shown. B, The amount of phosphatidylinositol phosphate [PI(P)] was quantitated by densitometry. Results are the mean ± SE of three separate experiments.

View larger version (19K): [in this window] [in a new window]   Figure 9. Effects of Shc overexpression on Akt activity. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h. IGF-1-induced Akt activity in the transfected cells was measured as described in Materials and Methods. A, Results of time course studies of 14 nM IGF-1-induced Akt activity are expressed as the mean ± SE of three separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: mock, b = 22,733 ± 1,923 dpm/1 x 106 cells, and m = 3.03 ± 0.28-fold; WT-Shc, b = 23,300 ± 1,892 dpm/1 x 106 cells, and m = 1.94 ± 0.19-fold; 3F-Shc, b = 22,320 ± 822 dpm/1 x 106 cells, and m = 2.14 ± 0.14-fold. B, Results of dose-response studies of IGF-1-induced Akt activity for 5 min are expressed as the mean ± SE of four separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: mock, b = 21,624 ± 1,042 dpm/1 x 106 cells, and m = 3.10 ± 0.27-fold; WT-Shc, b = 22,385 ± 1,226 dpm/1 x 106 cells, and m = 2.09 ± 0.21-fold; 3F-Shc, b = 21,921 ± 1,088 dpm/1 x 106 cells, and m = 2.16 ± 0.17-fold.

View larger version (26K): [in this window] [in a new window]   Figure 10. Effects of Shc overexpression on glucose incorporation into glycogen. L6 myoblasts overexpressing mock, WT-Shc, or 3F-Shc were serum-starved for 24 h. IGF-1-induced glucose incorporation into glycogen in the transfected cells was measured as described in Materials and Methods. Results are the mean ± SE of three separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: mock, b = 8838 ± 313 dpm/1 x 106 cells, and m = 2.63 ± 0.17-fold; WT-Shc, b = 7292 ± 297 dpm/1 x 106 cells, and m = 2.29 ± 0.28-fold; 3F-Shc, b = 7962 ± 317 dpm/1 x 106 cells, and m = 2.26 ± 0.11-fold.

View larger version (21K): [in this window] [in a new window]   Figure 11. Effects of PTB-Shc overexpression on Akt activity. L6 myoblasts overexpressing mock, WT-Shc, or PTB-Shc were serum-starved for 24 h. IGF-1-induced Akt kinase activity in the transfected cells was measured as described in Materials and Methods. Results are the mean ± SE of four separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: mock, b = 22,733 ± 1,923 dpm/1 x 106 cells, and m = 3.03 ± 0.28-fold; WT-Shc, b = 23,300 ± 1,892 dpm/1 x 106 cells, and m = 1.94 ± 0.19-fold; PTB-Shc, b = 23,029 ± 1,789 dpm/1 x 106 cells, and m = 1.99 ± 0.10-fold.

View larger version (26K): [in this window] [in a new window]   Figure 12. Effects of PTB-Shc overexpression on glucose incorporation into glycogen. L6 myoblasts overexpressing mock, WT-Shc, or PTB-Shc were serum-starved for 24 h. IGF-1-induced glucose incorporation into glycogen in the transfected cells was measured as described in Materials and Methods. Results are expressed as the mean ± SE of three separate experiments. Absolute counts of basal levels (b) and maximal stimulations (m) were as follows: mock, b = 8838 ± 313 dpm/1 x 106 cells, and m = 2.63 ± 0.17-fold; WT-Shc, b = 7292 ± 297 dpm/1 x 106 cells, and m = 2.29 ± 0.28-fold; 3F-Shc, b = 7013 ± 258 dpm/1 x 106 cells, and m = 2.15 ± 0.21-fold.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Shc Tyr-317 residue was originally characterized as a primary phosphorylation site of Shc (9, 11). However, recent studies have identified alternative Shc phosphorylation sites on Tyr-239/240 residues (36, 37, 38). Relative involvement of Shc Tyr-239/240 vs. Tyr-317 as the phosphorylation sites appears to be controversial, depending on ligands and cell types. Thus, we have shown that Shc Tyr-317 residue, rather than Shc Tyr-239/240 residues, is mainly involved in the coupling with Grb2 leading to MAPK activation in insulin signaling in Rat1 fibroblasts (26, 39). In contrast, phosphorylation on Shc Tyr-239/240 residues, but not on the Tyr-317 residue, is required for nerve growth factor-induced neurite outgrowth in PC12 cells (36). On the other hand, the relative role of Shc Tyr-239/240 residues vs. Tyr-317 for Grb2 binding leading to MAPK activation is controversial in EGF signaling (36, 37, 38). In IGF-1 signaling, it is unknown which tyrosine residue(s) of Shc are responsible for the phosphorylation and subsequent association with Grb2. Therefore, to unambiguously clarify the role of Shc tyrosine phosphorylation in IGF-1 signaling, we constructed a mutant Shc in which three possible tyrosine phosphorylation sites were all mutated to phenylalanine. As a result, exogenously transfected 3F-Shc was not detectably tyrosine-phosphorylated upon IGF-1 stimulation. In addition, IGF-1-stimulated tyrosine phosphorylation of endogenous Shc was decreased, whereas the amount of endogenous Shc expression was unchanged as shown in Figs. 1 and 2. These results indicate that the Shc mutant with Tyr-239/240/317-Phe residues (3F-Shc) functions as a dominant negative mutant to inhibit endogenous Shc in our experimental condition. Thus, the study of 3F-Shc cells has important implications for understanding the mechanisms of IGF-1-induced signal transduction through Shc tyrosine phosphorylation. Our results showed that IGF-1-induced Shc association with Grb2 and MAPK activation was decreased by overexpression of 3F-Shc in L6 myoblasts. IGF-1 stimulation of thymidine incorporation was also decreased in 3F-Shc cells compared with that in parental L6 cells. These results indicate the essential role of Shc tyrosine phosphorylation in IGF-1 signaling leading to cell cycle progression, which is consistent with recent reports showing that Shc tyrosine phosphorylation plays a critical role in insulin-induced mitogenesis (12, 26).

Although the cellular mechanisms that regulate IGF-1-induced glycogen synthesis remain unknown, IGF-1 stimulation of glycogen synthesis is known to be regulated by the activation of glycogen synthase via its dephosphorylation (23, 24, 28). Because activation of glycogen synthase can be mediated by activation of protein phosphatase 1, which dephosphorylates glycogen synthase (6), and MAPK-p90rsk pathway locates, at least in vitro, upstream of protein phosphatase 1 (39), original studies indicated the possible involvement of MAPK in the activation of glycogen synthesis. However, increasing evidence suggests that activation of glycogen synthase is mediated by the ß-isoforms of glycogen synthase kinase 3 in L6 cells (23, 24, 28). Because the ß-isoform of glycogen synthase kinase 3 is regulated by Akt, which lies downstream of PI3K (23, 24), IGF-1-induced PI3K activation appears to be important for the glycogen synthesis. In this regard, our results showed that overexpression of WT-Shc into L6 myoblasts led to decreased IGF-1 stimulation of glucose incorporation into glycogen compared with that in parental L6 cells. Importantly, IGF-1-induced glucose incorporation into glycogen was also reduced by overexpression of 3F-Shc. It is possible that much greater inhibition of IGF-1-induced glycogen synthesis, including apparent reduction of responsiveness, may be seen if the transfection efficiency of these Shc constructs is higher. Although the degree of inhibition by the transfection remains to be clarified, these results clearly indicate that, consistent with the recent understanding, MAPK-mediated pathway is not involved in the activation of IGF-1-induced glycogen synthesis and Shc negatively regulates glycogen synthesis by the mechanism that tyrosine phosphorylation of Shc is not relevant.

It is interesting to speculate about the mechanism(s) by which Shc negatively regulates glycogen synthesis. Shc and IRS-1 are the major intracellular substrates of the activated IGF-1 receptor (34). IRS-1 is found to interact with the juxtamembrane domain around Tyr-950 of the activated IGF-1 receptor (34). Shc has also been reported to interact with the same region of the IGF-1 receptor (34). Therefore, one can speculate that Shc and IRS-1 bind competitively to phosphorylated IGF-1 receptors. Along this line, our results demonstrated that the amount of tyrosine phosphorylation of IRS-1 was decreased in WT-Shc and 3F-Shc as shown in Fig. 6. Thus, our results indicated that transiently overexpressed Shc could compete with endogenous IRS-1 for the interaction with IGF-1 receptors in L6 cells. In addition, overexpression of 3F-Shc as well as WT-Shc also decreased IGF-1 stimulated IRS-1 phosphorylation. These results clearly showed that the competitive interaction of Shc and IRS-1 with the IGF-1 receptor by the mechanism that tyrosine phosphorylation of Shc is not relevant. This is consistent with the previous reports showing that the competitive interaction of Shc and IRS-1 with the insulin receptor is seen (25, 26, 40). As mentioned above, PI3K is known to be an important molecule for the signal transduction leading to glycogen synthesis (23, 24, 28). Because IGF-1-induced activation of PI3K is mediated by coupling of tyrosine-phosphorylated IRS-1 with the p85 regulatory subunit of PI3K (16), IRS-1 appears to be a key molecule for IGF-1-induced glycogen synthesis. This idea was further supported by the fact that overexpression of WT-Shc decreased IGF-1 stimulation of IRS-1 association with the p85 subunit of PI3K and of PI3K activity, and that the reduction was proportional to the decrease of tyrosine phosphorylation of IRS-1. Furthermore, as the competitive mechanism of Shc for IRS-1 interaction with the IGF-1 receptor, both IRS-1 and Shc, via these PTB domains, interact with the juxtamembrane domain of the activated IGF-1 receptor via the NPEY motif surrounding tyrosine residue 950 (34). Along this line, it can be speculated that competitive interaction via the PTB domain of Shc regulates biological action of IGF-1. Our results showed that overexpression of Shc PTB domain alone decreased IGF-1-induced Akt activation and glucose incorporation into glycogen compared with those in parental L6 myoblasts. These results further indicate that decreased signaling pathways leading to glycogen synthesis by overexpression of both WT-Shc and 3F-Shc largely appear to depend on the inhibitory effect of IRS-1 mediated signaling pathway by the competitive interaction via the PTB domain of Shc.

In summary, our results indicate that tyrosine phosphorylation of Shc is important for mediating MAPK activation leading to cell cycle progression. In contrast, Shc appears to negatively regulate IGF-1-induced glycogen synthesis, at least in part, by down-regulating the PI3K-Akt pathways. Because both Shc and IRS-1 are substrates for the IGF-1 receptor, Shc competes with IRS-1 to be phosphorylated is a possible mechanism of the inhibitory role. In this regard, in contrast to the key role of Shc tyrosine phosphorylation in IGF-1-mediated mitogenic signaling, only the Shc PTB domain is required, and Shc tyrosine phosphorylation is not relevant for inhibition of the IRS-1-mediated signaling leading to glycogen synthesis.

	Acknowledgments

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

	Footnotes

 
This work was supported in part by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (to T.S.).

¹ These two authors contributed equally to this work.

Abbreviations: 3F, Y239,240,317F; CH, collagen homology; DTT, dithiothreitol; EGF, epidermal growth factor; IRS, insulin receptor substrate; PMSF, phenylmethylsulfonyl fluoride; PTB, phosphotyrosine binding; WT, wild-type.

Received April 30, 2001.

Accepted for publication August 16, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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