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. 2008 Sep;22(9):2162–2175. doi: 10.1210/me.2008-0079

p66shc Negatively Regulates Insulin-Like Growth Factor I Signal Transduction via Inhibition of p52shc Binding to Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 Leading to Impaired Growth Factor Receptor-Bound Protein-2 Membrane Recruitment

Gang Xi 1, Xinchun Shen 1, David R Clemmons 1
PMCID: PMC2631373  PMID: 18606861

Abstract

Our previous studies have indicated an essential role of p52shc in mediating IGF-I activation of MAPK in smooth muscle cells (SMC). However, the role of the p66 isoform of shc in IGF-I signal transduction is unclear. In the current study, two approaches were employed to investigate the role of p66shc in mediating IGF-I signaling. Knockdown p66shc by small interfering RNA enhanced IGF-I-stimulated p52shc tyrosine phosphorylation and growth factor receptor-bound protein-2 (Grb2) association, resulting in increased IGF-I-dependent MAPK activation. This was associated with enhanced IGF-I-stimulated cell proliferation. In contrast, knockdown of p66shc did not affect IGF-I-stimulated IGF-I receptor tyrosine phosphorylation. Overexpression of p66shc impaired IGF-I-stimulated p52shc tyrosine phosphorylation and p52shc-Grb2 association. In addition, IGF-I-dependent MAPK activation was also impaired, and SMC proliferation in response to IGF-I was inhibited. IGF-I-dependent cell migration was enhanced by p66shc knockdown and attenuated by p66shc overexpression. Mechanistic studies indicated that p66shc inhibited IGF-I signal transduction via competitively inhibiting the binding of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to SHP substrate-1 (SHPS-1), leading to the disruption of SHPS-1/SHP-2/Src/p52shc complex formation, an event that has been shown previously to be essential for p52shc phosphorylation and Grb2 recruitment. These findings indicate that p66shc functions to negatively regulate the formation of a signaling complex that is required for p52shc activation in response to IGF-I, thus leading to attenuation of IGF-I-stimulated cell proliferation and migration.

SRC HOMOLOGY (Shc) protein is an adaptor protein that mediates signal transduction linking multiple tyrosine kinase growth factor receptors to activation of the MAPK pathway. Three shc genes have been identified, shcA, shcB, and shcC (1). ShcA has been shown to be widely expressed in human and mouse tissues except mature neurons, which utilize shcC (2,3). ShcB is primarily expressed in the brain but its function has not been well characterized (1). ShcA (hereafter referred to as shc) is expressed as three isoforms with molecular masses of 46, 52, and 66 kDa. These three isoforms arise as a result of alternative translational initiation sites and mRNA splicing (2,4). Previous studies have shown that p52 and p46shc are phosphorylated on tyrosine 239, 240, and 317 after growth factor stimulation. These phosphotyrosines provide binding sites for growth factor receptor-bound protein-2 (Grb2), which activates Ras, leading to MAPK activation (5,6,7,8). Grb2 binding to shc is required for IGF-I to stimulate growth in a variety of cell types, including 3T3-L1 preadipocytes (9), CHO cells (7,10), and neuroblastoma cells (11). In smooth muscle cells (SMC), p52shc tyrosine phosphorylation and Grb2 binding have been shown to be necessary for mediating IGF-I signal transduction. Impairment of p52shc activation either by overexpression of a Y239, 240, and 317F (shc-3F) mutant shc (12) or by blocking activation of the upstream kinase, Src, using small interfering RNA (13) attenuated the IGF-I-stimulated MAPK phosphorylation and cell proliferation. Similar to p52/p46shc, p66shc also contains these three critical tyrosines, and they are phosphorylated after growth factor stimulation. Epidermal growth factor (EGF) has been shown to stimulate p66shc tyrosine phosphorylation, but in contrast to p52shc activation, c-fos promoter activation is inhibited and there is no stimulation of MAPK activation (4). In addition, p66shc was reported to negatively regulate EGF-stimulated MAPK activation, but the mechanism that mediated this effect was not elucidated (14).

In addition to tyrosine phosphorylation, p66shc contains multiple serine/threonine phosphorylation sites. Among them, the Ser36 site is located within the unique CH2 domain. Previous studies have shown that Ser36 phosphorylation of p66shc is responsible for limiting mouse lifespan extension (15) and mediating intracellular reactive oxygen species regulation of forkhead proteins (16) as well as amyloid β-peptide toxicity in Alzheimer’s disease (17). The extended lifespan of p66shc null mice has been linked to a decreased mitochondrial metabolism (18) and reactive oxygen species production (19). Most recently, protein kinase Cβ was reported to be activated by oxidative stress and to induce Ser36 phosphorylation of p66shc, resulting in mitochondrial accumulation of p66shc and alterations in mitochondrial Ca2+ responses and three-dimensional structure. These changes were attributed to enhanced apoptosis (20).

In contrast to the well-defined role of p66shc in mediating the cellular response to oxidative stress, the role of p66shc in mediating signaling after growth factor stimulation has not been determined. Our previous studies have shown that the p52shc activation is required for IGF-I-stimulated MAPK activation and cell proliferation in SMC (12,13). In the present study, we used the molecular approaches to induce both loss and gain of function to determine the effect of p66shc on IGF-I-dependent signaling events that are proximal and downstream of p52shc activation.

RESULTS

Knockdown of p66shc Significantly Enhances IGF-I-Dependent p52shc Tyrosine Phosphorylation and Grb2 Association

Our previous studies have demonstrated the importance of p52shc tyrosine phosphorylation for mediating IGF-I signal transduction in porcine SMC (pSMC) (12,13). However, hyperglycemia, which induces oxidative stress, a condition known to activate p66shc, enhances SMC responses to IGF-I (21). To more precisely determine the relative roles of p66 and p52shc in mediating IGF-I actions, we determined the effect of p66shc knockdown on IGF-I-dependent p52shc phosphorylation. After RNA interference (RNAi) transfection, the p66shc protein level was decreased 87 ± 2% (P < 0.01; n = 3) compared with empty vector control (EVC) pSMC, whereas the protein levels of p52shc and p46shc showed no significant change (Fig. 1A). When p52shc phosphorylation was analyzed, IGF-I stimulated a significantly greater increase in p52shc phosphorylation in the p66shc knockdown cells compared with EVC cells (e.g. 2.27 ± 0.10-fold increase vs. 1.43 ± 0.16-fold increase above basal level after 10 min treatment, P < 0.05; n = 3) (Fig. 1B). IGF-I-dependent p66shc tyrosine phosphorylation could be detected in EVC cells but not in the p66shc knockdown cells.

Figure 1.

Figure 1

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Knockdown of p66shc Enhances IGF-I-Dependent p52shc Tyrosine Phosphorylation and Grb2 Association

A, pSMC were transduced with empty vector or the p66shc shRNA template plasmid and analyzed for shc protein expression. Cell lysates were immunoblotted with anti-shc antibody. The arrows denote the p66shc, p52shc, and p46shc bands. B, Quiescent pSMC were stimulated with IGF-I (100 ng/ml) for the indicated times. Shc phosphorylation was determined by immunoprecipitating cell lysates with an anti-shc antibody and then immunoblotting with anti-p-Tyr (PY99) antibody. To control for loading, the blot was reprobed with an anti-shc antibody. The arrows denote the p66shc and p52shc bands. The protein levels were quantified using scanning densitometry. Each point represents the phosphorylated shc value divided by total shc and is the pool of at least three independent experiments, expressed as the mean ± sem. *, P < 0.05 indicates a significant difference at 10 and 20 min after IGF-I stimulation in p66shc knockdown cells compared with EVC cells. C, Cells were serum starved for 16–18 h and then stimulated with IGF-I (100 ng/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-Grb2 and immunoblotted with anti-shc. To control the loading, the blot was stripped and reprobed for anti-Grb2. The relative amount of p52shc associated with Grb2 was quantified using scanning densitometry, and the results were normalized by the Grb2 protein level as was done previously. *, P < 0.05 indicates that knockdown p66shc significantly enhances IGF-I-stimulated p52shc bound to Grb2, compared with control cells.

Previous studies have shown that tyrosine phosphorylation of p52shc provides a binding site for Grb2, which leads to IGF-I-stimulated Ras and MAPK activation (5,22,23). Therefore, we determined the effect of p66shc knockdown on the ability of p52shc to recruit Grb2. The results showed that knockdown p66shc significantly enhanced Grb2 association with p52shc after IGF-I stimulation (Fig. 1C). After 5 min IGF-I treatment, the amount of Grb2 bound to p52shc was increased 2.72 ± 0.28-fold above the basal level in p66shc knockdown cells, whereas a 1.97 ± 0.15-fold increase was detected in EVC cells (P < 0.05, p66shc knockdown vs. EVC cells). Similarly, IGF-I- dependent p66shc association with Grb2 was detected in EVC cells but not in the p66shc knockdown cells (Fig. 1C). These data suggest that p66shc functions to inhibit p52shc tyrosine phosphorylation and its ability to recruit the Grb2.

Knockdown of p66shc Does Not Alter IGF-I- Dependent IGF-I Receptor (IGF-IR) Activation

To exclude the possibility that p66shc was functioning to inhibit IGF-I signaling by directly altering activation of the IGF-IR kinase, we investigated the effect of p66shc knockdown on IGF-I-dependent IGF-IR tyrosine phosphorylation, which has been shown the essential for mediating IGF-I signal transduction in pSMC (24). The analysis indicated that there was no obvious difference of IGF-I-dependent IGF-IR tyrosine phosphorylation between p66shc knockdown cells and EVC cells (Fig. 2). IGF-I stimulated a 25.9 ± 0.7-fold increase in IGF-IR tyrosine phosphorylation in EVC cells, and there was a 26.8 ± 1.7-fold increase in the p66shc knockdown cells after 5 min IGF-I treatment (P not significant).

Figure 2.

Figure 2

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Knockdown of p66shc Does Not Change IGF-I-Stimulated IGF-IR Tyrosine Phosphorylation

Cells were serum starved for 16–18 h before the addition of IGF-I (100 ng/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-IGF-IR antibody and immunoblotted with anti-p-Tyr (PY99) antibody. To control the loading, the blot was stripped and reprobed with anti-IGF-IR antibody. The figure is representative of three independent experiments.

Overexpression of p66shc Impairs IGF-I- Dependent p52shc Tyrosine Phosphorylation and Its Ability to Recruit Grb2

To confirm these results using a different methodology, we used p66shc overexpression. Enhanced expression was verified by the detection of exogenous p66shc protein containing the hemagglutinin (HA) epitope (Fig. 3A). We then determined the effect of p66shc overexpression on IGF-I-dependent p52shc tyrosine phosphorylation. Consistent with our knockdown results, overexpression of p66shc significantly attenuated IGF-I-stimulated p52shc phosphorylation, e.g. a 1.89 ± 0.10-fold increase above basal level after 10 min of IGF-I treatment in LacZ control (LacZ) cells vs. a 1.28 ± 0.17-fold increase in p66shc-overexpressing cells (P < 0.05) (Fig. 3B). An increase in IGF-I-dependent p66shc phosphorylation was also observed in p66shc overexpressing cells (Fig. 3B). Similarly, overexpression of p66shc reduced the ability of p52shc to recruit Grb2 by 32 ± 4% (P < 0.05), 66 ± 12% (P < 0.05), and 71 ± 17% (P < 0.05) at 5, 10, and 20 min after IGF-I treatment, respectively, compared with LacZ cells (Fig. 3C). To exclude the possibility that these changes were caused by the alteration of IGF-IR activation after p66shc overexpression, we quantified IGF-IR tyrosine phosphorylation. No difference in IGF-I-dependent IGF-IR tyrosine phosphorylation was detected when p66shc-overexpressing cells and LacZ cells were compared (data not shown). In addition, greater p66shc association with Grb2 was observed after IGF-I stimulation in p66shc-overexpressing cells. These data provide further evidence that p66shc normally inhibits IGF-I-dependent p52shc tyrosine phosphorylation and Grb2 association.

Figure 3.

Figure 3

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Overexpression of p66shc Impairs IGF-I-Stimulated p52shc Tyrosine Phosphorylation and Grb2 Association

A, pSMC were transducted with pLenti-LacZ or pLenti-HA p66shc vector. Both cell types were serum starved for 16–18 h and analyzed for p66shc and HA protein expression. Cell lysates were immunoblotted with anti-shc or anti-HA antibodies. The arrows denote the exogenous (Exo) and endogenous (Endo) p66shc, p52shc, and p46shc bands. B, Quiescent pSMC were stimulated with IGF-I (100 ng/ml) at indicated times. Shc phosphorylation was determined by immunoprecipitating cell lysates with an anti-shc antibody and then immunoblotting with an anti-p-Tyr (PY99) antibody. For a loading control, the blot was reprobed with anti-shc antibody. The arrows denote the p66 and p52shc bands. The protein levels were quantified using scanning densitometry. Each point represents the phosphorylated shc value divided by total shc and is the pool of at least three independent experiments, expressed as the mean ± sem. *, P < 0.05 indicates a significant difference at 5, 10, and 20 min after IGF-I stimulation in p66shc overexpression cells compared with LacZ control cells. C, Cells were serum starved for 16–18 h before the addition of IGF-I (100 ng/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-Grb2 polyclonal antibody and immunoblotted with anti-shc antibody. To control the loading, the blot was stripped and reprobed with anti-Grb2 monoclonal antibody. The arrows denote the p66 and p52shc bands. The figure is representative of three independent experiments. The relative amount of p52shc associated with Grb2 was quantified using scanning densitometry, and the results were normalized by the Grb2 protein level. *, P < 0.05 indicates that overexpression of p66shc significantly impairs IGF-I-stimulated p52shc binding to Grb2, compared with LacZ control cells.

IGF-I-Stimulated MAPK Activation Is Enhanced by Knockdown of p66shc and Impaired by Its Overexpression

Because knockdown and overexpression of p66shc significantly changed IGF-I-dependent p52shc phosphorylation and Grb2 association, we further investigated the effect of p66shc on IGF-I-stimulated MAPK activation. The analysis showed that knockdown of p66shc was associated with a significant increase in IGF-I-dependent Erk1/2 phosphorylation compared with EVC cells (e.g. a 1.60 ± 0.12-fold greater increase compared with EVC cells at 10 min after IGF-I treatment, P < 0.05) (Fig. 4A). Of note, knockdown of p66shc resulted in sustained MAPK activation. After 20 min IGF-I stimulation, the relative Erk1/2 activation was only 76 ± 10% above the basal level in EVC cells, but it was maintained at 188 ± 12% above the basal level in the p66shc knockdown cells (P < 0.001, compared with the response of EVC cells) (Fig. 4A). Consistent with p66shc knockdown data, overexpression p66shc in pSMC significantly impaired IGF-I-dependent MAPK activation (Fig. 4B). Specifically, in LacZ and p66shc-overexpressing cells, Erk1/2 phosphorylation was increased by 7.19 ± 0.87- vs. 4.89 ± 0.58-fold (P < 0.05), 7.76 ± 0.59- vs. 5.11 ± 0.66-fold (P < 0.05) and 3.39 ± 0.58- vs. 1.71 ± 0.43-fold (P < 0.05) above basal level at 5, 10, and 20 min after IGF-I treatment, respectively. These data show that p66shc inhibits IGF-I-stimulated downstream signaling in pSMC. In addition, we also examined the effect of p66shc on p38MAPK and c-Jun N-terminal kinase (JNK) activation after IGF-I stimulation. The results showed no significant differences were detected in p66shc-overexpressing or knockdown cells, compared with control cells, respectively (data not shown).

Figure 4.

Figure 4

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p66shc Negatively Regulates IGF-I-Stimulated MAPK Activation in pSMC

After IGF-I (100 ng/ml) treatment for the indicated times, cell lysates from EVC and p66shc shRNA template (p66shc Si) (A) or pLenti-LacZ (LacZ) and pLenti-HA p66shc (p66shc O) vector (B) transduced cells were used to determine the activation of Erk1/2 by probing with anti-phospho-Erk1/2 antibody. The blots were stripped and reprobed with an anti-Erk1/2 antibody to control for loading differences. The phosphorylation of Erk1/2 normalized by the protein levels was quantified using scanning densitometry. Each point is the mean of at least three independent experiments and is indicated as mean ± sem. *, P < 0.05; ***, P < 0.001 indicates a significant difference between IGF-I-stimulated activation of Erk1/2 in p66shc knockdown or overexpressing cells compared with control cells, respectively.

p66shc Negatively Regulates IGF-I-Stimulated Cell Proliferation and Migration

Because we showed that IGF-I signaling was significantly enhanced by p66shc knockdown and impaired by its overexpression, we further investigated whether these changes would affect IGF-I-stimulated biological functions. To control for nonspecific effects, we determined whether knockdown p66shc would influence SMC growth response to serum. The results indicated that both cell types responded to serum with a 2- to 3-fold increase in cell number after 48 h, which was comparable to nontransfected pSMC cultures (Fig. 5A). Next we compared IGF-I-dependent cell proliferation in all three cell types. The results showed that IGF-I-stimulated cell proliferation was increased in p66shc knockdown cells (1.87 ± 0.09-fold increase vs. 1.43 ± 0.07-fold increase above basal level, P < 0.01, compared with EVC cells), and it was decreased in p66shc-overexpressing cells (1.16 ± 0.01-fold increase vs. 1.37 ± 0.01-fold increase above basal level, P < 0.01, compared with LacZ cells) (Fig. 5B). We have previously reported that IGF-I stimulates pSMC migration (25). Therefore we examined the effect of p66shc knockdown on IGF-I-dependent cell migration. The results indicated that IGF-I-stimulated cell migration was increased 2.14 ± 0.11-fold above basal level in p66shc knockdown cells, compared with a 1.66 ± 0.05-fold increase above the basal level in EVC cells (P < 0.05, p66shc knockdown cells vs. EVC cells) (Fig. 5C). In addition, cell migration was impaired by overexpression of p66shc (1.71 ± 0.06- vs. 1.41 ± 0.03-fold above basal level after IGF-I stimulation, P < 0.05) (Fig. 5C). Consistent with the alterations in IGF-I signaling, the findings clearly demonstrate that p66shc functions to negatively regulate IGF-I-stimulated cell proliferation and migration in pSMC.

Figure 5.

Figure 5

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p66shc Negatively Regulates IGF-I-Stimulated Proliferation and Migration in pSMC

Cell proliferation and migration were determined as described in Materials and Methods. A, Comparison of the cell proliferative response in serum containing medium between p66shc knockdown (p66shc Si) and control (p66shc Wt) cells. B, Comparison of the cell proliferative response to IGF-I between p66shc knockdown (p66shc Si) and control cells (EVC) and p66shc-overexpressing (p66shc O) and control cells (LacZ). Each bar indicates the fold increase over basal and represents the pool of at least three independent experiments. P < 0.01 indicates that knockdown p66shc significantly enhances IGF-I-dependent cell proliferation and overexpression of p66shc significantly impairs IGF-I-stimulated cell proliferation, compared with control cells, respectively. C, Comparison of IGF-I-stimulated cell migration between p66shc knockdown (p66shc Si) and control cells (EVC) and p66shc-overexpressing (p66shc O) and control cells (LacZ), respectively. Each bar indicates the fold increase over basal and represents the pool of at least three independent experiments. P < 0.05 indicates that p66shc knockdown significantly enhances IGF-I-dependent cell migration and overexpression of p66shc significantly attenuates it, compared with control cells, respectively.

p66shc Inhibits IGF-I Signal Transduction by Competitively Binding to Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 (SHPS-1) and Preventing Assembly of the SHP-2/Src/p52shc/Grb2 Signaling Complex

Our previous studies have shown that p52shc must be recruited to the cell surface-associated protein SHPS-1 before it can be tyrosine phosphorylated (12,13). Therefore we determined the effect of knockdown and overexpression of p66shc on IGF-I-dependent SHPS-1 phosphorylation and on the formation of the SHPS-1/p52shc/Grb2 complex. The results showed that knockdown or overexpression of p66shc did not affect IGF-I-dependent SHPS-1 phosphorylation (Figs. 6A and 7A). However, IGF-I-dependent p52shc association with SHPS-1 was enhanced in p66shc knockdown cells (Fig. 6A) and impaired in p66shc-overexpressing cells (Fig. 7A). In addition, the results showed overexpression of p66shc led to enhanced p66shc binding to SHPS-1, compared with LacZ cells (Fig. 7A). Knockdown of p66shc enhanced IGF-I-stimulated SHPS-1/SHP-2 association at 5 min after IGF-I stimulation (Fig. 6B), whereas overexpression of p66shc greatly impaired SHPS-1/SHP-2 association (Fig. 7B). Because SHP-2 binding to SHPS-1 as well as Src binding to SHP-2 have been shown to be essential for Src and p52shc association with SHPS-1 (13), we examined Src/SHPS-1 and Src/p52shc association. Disruption of the SHP-2/SHPS-1 complex was associated with a reduction in Src and SHPS-1 as well as Src and p52shc association (Fig. 7, C and D). Consistently, knockdown of p66shc enhanced this signal complex formation after IGF-I stimulation (Fig. 6, C and D). To compare the differences of SHPS-1/SHP-2/Src complex formation in response to IGF-I between p66shc knockdown and p66shc-overexpressing cells, we quantified the changes in this complex formation after IGF-I stimulation in both systems. For Src complex formation (Figs. 6C and 7C), SHPS-1 association with Src was increased by 2.13 ± 0.20- and 4.02 ± 0.18-fold (P < 0.001) in EVC cells and p66shc knockdown cells, respectively, after 5 min IGF-I treatment, whereas this association was increased by 1.97 ± 0.16- and 1.25 ± 0.03-fold (P < 0.05) in LacZ cells and p66shc-overexpressing cells after 10 min IGF-I treatment. SHP-2 association with Src was increased by 1.81± 0.20 and 2.68 ± 0.20-fold (P < 0.05) after 5 min IGF-I stimulation in EVC cells and p66shc knockdown cells, whereas this association was increased by 2.44 ± 0.25 and 1.36 ± 0.19 (P < 0.05) in LacZ cells and p66shc-overexpressing cells. For SHP-2 complex formation (Figs. 6D and 7D), SHPS-1 association with SHP-2 was increased by 1.66 ± 0.06- and 2.05 ± 0.10-fold (P < 0.05) in EVC cells and p66shc knockdown cells, respectively, after 5 min IGF-I treatment, whereas this association was increased 2.14 ± 0.25- and 1.46 ± 0.24-fold (P < 0.05) in LacZ cells and p66shc-overexpressing cells after 10 min IGF-I treatment. Src association with SHP-2 was increased by 1.83 ± 0.06- and 2.85 ± 0.10-fold (P < 0.01) after 5 min IGF-I stimulation in EVC cells and p66shc knockdown cells, whereas this association was increased by 3.46 ± 0.29 and 2.11 ± 0.17 (P < 0.05) in LacZ cells and p66shc-overexpressing cells. In addition, the association of both Src and p52shc with SHPS-1 appears to be indirect and is mediated through their binding to SHP-2 (13); therefore, by inhibiting SHP-2 association with SHPS-1, p66shc functions to inhibit SHP-2/Src/p52shc complex formation and p52shc phosphorylation. Interestingly, in addition to increased binding to SHPS-1, there was also increased p66shc association with Src and SHP-2 after IGF-I stimulation in p66shc-overexpressing cells (Fig. 7, C and D).

Figure 6.

Figure 6

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Knockdown of p66shc Enhances IGF-I-Stimulated SHPS-1/SHP-2/Src/p52shc/Grb2 Complex Formation in pSMC

Quiescent pSMC were stimulated with IGF-I (100 ng/ml) for the indicated times. A, The cell lysates were immunoprecipitated with anti-SHPS-1 polyclonal antibody and immunoblotted with anti-p-Tyr (PY99), -shc, and -Grb2 antibody, respectively. To control for loading, the blot was stripped and immunoblotted with anti-SHPS-1 antibody. B, The cell lysates were immunoprecipitated with anti-SHPS-1 polyclonal antibody and immunoblotted with anti-SHP-2 and c-Src antibodies, respectively. To control for loading, the blot was stripped and immunoblotted with anti-SHPS-1 antibody. C, The cell lysates were immunoprecipitated with anti-c-Src antibody and immunoblotted with anti-SHPS-1, -SHP-2, and -shc antibody, respectively. As a loading control, the blot was stripped and immunoblotted with anti-c-Src antibody. D, The cell lysates were immunoprecipitated with anti-SHP-2 antibody and immunoblotted with anti-SHPS-1, -shc, and -Src antibodies, respectively. As a loading control, the blot was stripped and immunoblotted with anti-SHP-2 antibody. The figures are representative of three independent experiments.

Figure 7.

Figure 7

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Overexpression p66shc Impairs IGF-I-Stimulated SHPS-1/SHP-2/Src/p52shc/Grb2 Complex Formation in pSMC

Quiescent pSMC were stimulated with IGF-I (100 ng/ml) for the indicated times. A, The cell lysates were immunoprecipitated with anti-SHPS-1 polyclonal antibody and immunoblotted with anti-p-Tyr (PY99), -shc, and -Grb2 antibodies, respectively. For a loading control, the blot was stripped and immunoblotted with anti-SHPS-1 antibody. B, The cell lysates were immunoprecipitated with anti-SHPS-1 polyclonal antibody and immunoblotted with anti-SHP-2 and -c-Src antibodies, respectively. To control for loading, the blot was stripped and immunoblotted with anti-SHPS-1 antibody. C, The cell lysates were immunoprecipitated with anti-c-Src antibody and immunoblotted with anti-SHPS-1, -SHP-2, and -shc antibody, respectively. As a loading control, the blot was stripped and immunoblotted with anti-c-Src antibody. D, The cell lysates were immunoprecipitated with anti-SHP-2 antibody and immunoblotted with anti-SHPS-1, -shc, and -c-Src antibody, respectively. As a loading control, the blot was stripped and immunoblotted with anti-SHP-2 antibody. The figures are representative of three independent experiments.

p66shc Inhibits IGF-I Signal Transduction via Impairing Membrane Recruitment of Grb2

To further elucidate the function of p66shc, we isolated the cytoplasmic and membrane fractions from p66shc-overexpressing and LacZ cells. Although there was no difference in total Grb2 protein in the cytoplasmic fraction before and after IGF-I stimulation in p66shc-overexpressing cells (Fig. 8, A and C), it was significantly decreased after IGF-I stimulation in LacZ cells (Fig. 8, A and C). In addition, increases of total Grb2 and p52shc proteins were detected in the membrane fraction in LacZ cells after IGF-I stimulation but were not detected in p66shc-overexpressing cells (Fig. 8, A and C). This is consistent with the results in Fig. 7A, which showed no increase of SHPS-1/p52shc/Grb2 complex formation in p66shc-overexpressing cells after IGF-I stimulation. To confirm the validity of the cell fractionation procedure, caveolin and 14-3-3β were used as the marker proteins for membrane and cytoplasmic fractions, respectively (Fig. 7B). Taken together, these data suggest that p66shc negatively regulates IGF-I signal transduction by inhibiting p52shc association with SHPS-1, the site of p52shc phosphorylation, and thus preventing Grb2 recruitment to the membrane, which is necessary for IGF-I-stimulated downstream signaling.

Figure 8.

Figure 8

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Overexpression of p66shc Impairs IGF-I-Stimulated p52shc and Grb2 Membrane Recruitment in pSMC

Quiescent pSMC were stimulated with IGF-I for 10 min and fractionated as described in Materials and Methods. A, Equal amounts of cytoplasmic and membrane fraction protein were analyzed for shc or Grb2 by immunoblotting. B, To confirm the validity of the cell fractionation procedure, equal amount of protein from the different fractions were analyzed to determine the presence of marker proteins for membrane (anti-caveolin) and cytoplasmic (anti-14-3-3β) fractions, respectively. The figures are representative of three independent experiments. C, Comparison of total Grb2 protein distribution after IGF-I stimulation between p66shc-overexpressing (p66shc O) cells and control (LacZ) cells. Each bar indicates the relative amount of Grb2 (Grb2 protein/marker protein) in each fraction and represents the pool of three independent experiments. P < 0.05 and P < 0.01 indicate IGF-I stimulation significantly decreases Grb2 protein in cytoplasmic fraction and increases Grb2 protein to be recruited to membrane fraction in control cells.

DISCUSSION

The p52 isoform of shc undergoes tyrosine phosphorylation after growth factor stimulation, which results in Grb2 recruitment and subsequent Ras and MAPK activation (2,26,27,28,29). Our previous studies have demonstrated that p52shc tyrosine phosphorylation is required for IGF-I to stimulate MAPK activation and mitogenesis in pSMC (12,13). In contrast, the role of the p66 isoform of shc, which is also phosphorylated in response to IGF-I in mediating IGF-I-stimulated signaling events and biological actions, has not been defined. The results obtained in this study using the p66shc knockdown or its overexpression lead us to conclude that p66shc inhibits IGF-I-stimulated p52shc tyrosine phosphorylation and Grb2 recruitment as well as MAPK activation, thus resulting in a decrease in IGF-I-stimulated proliferation and migration.

Our experiments to determine the mechanism by which p66shc functions to inhibit IGF-I actions focused on the role of SHPS-1. SHPS-1 is a transmembrane protein that plays a major role in mediating various cellular responses. The cytoplasmic domain of SHPS-1 contains four tyrosine residues located within YXXL/I/V motifs, which are phosphorylated after growth factor stimulation or cell adhesion and then bind to SH2 domain-containing proteins, such as SHP-2 (30,31,32). The transfer of SHP-2 to SHPS-1 is a required event for growth factor-mediated cell proliferation, DNA synthesis, shc phosphorylation, and MAPK activation (12,13,31,33,34). Our previous studies showed that disruption of SHP-2 binding to SHPS-1 either by inhibiting SHPS-1 phosphorylation (34) or using a blocking peptide (12) resulted in impairment of sustained MAPK activation. SHPS-1 acts as a scaffold protein providing docking sites for tyrosine phosphatases (31,35), Src family kinases (13,36), and adaptor proteins, such as Grb2 (35) and p52shc (12,13). We recently reported that the recruitment of p52shc to SHPS-1 is required for IGF-I-dependent p52shc tyrosine phosphorylation and subsequent downstream signaling (12). We showed that a complex assembles on SHPS-1 that contains SHP-2, Src, and p52shc. Src phosphorylates p52shc, and disruption of Src/shc or SHP-2/Src impairs IGF-I-dependent p52shc tyrosine phosphorylation and MAPK activation (13). In this study, we found that overexpression of p66shc impaired SHP-2 association with SHPS-1 in response to IGF-I, whereas IGF-I-stimulated SHPS-1 tyrosine phosphorylation status was not affected. In addition, there was increased association of p66shc with SHPS-1, suggesting that it was competing with SHP-2 for binding to SHPS-1. To further determine the etiology, we analyzed p52shc association with Src and Src association with SHP-2. Overexpression of p66shc resulted in inhibition of Src and p52 shc association with SHPS-1. Because our previous studies had shown that p52shc and Src association required SHP-2 association with SHPS-1, we predicted that inhibition of SHP-2 binding would also disrupt p52shc and Src association with the signaling complex, thus leading to decreased p52shc tyrosine phosphorylation. This prediction was confirmed. In addition, all of the IGF-I signal transduction events were enhanced in p66shc knockdown cells, supporting the conclusion that the levels of p66shc that are present in SMC are sufficient to negatively regulate IGF-I signaling. Of note, a small change of p66shc association with SHPS-1 leads to a significant change of p52shc in the SHPS-1 complex in response to IGF-I stimulation. This implies that, in addition to stoichiometric competition, p66shc may change IGF-I-dependent Src kinase activation, which could regulate both p52shc phosphorylation and SHPS-1 association.

Previous studies have shown that the subcellular localization of shc isoforms is an essential aspect of signal transduction (37,38,39,40). All three isoforms of shc have been reported to reside predominantly in the cytoplasm, and some redistribute to the plasma membrane after growth factor stimulation (37). Importantly, targeting p52shc to membrane rafts has been shown to constitutively activate the Ras/MAPK pathway (38). Our current cell fractionation data are consistent with those results and show that a large amount of p66shc and p52/p46shc are present in the cytoplasmic fraction, and the primary shc isoform, which is recruited to the membrane fraction after IGF-I stimulation, is the p52/p46 isoform. Previous studies indicate that subcellular localization of Grb2 is also critical for its ability to couple tyrosine kinase receptor signaling to Ras activation (41,42,43,44). Once Grb2 is recruited to the plasma membrane, primarily via binding with tyrosine-phosphorylated p52shc (45,46,47), it provides a docking site for son of sevenless (SOS), which leads to Ras and MAPK activation (48,49,50,51). Our results which show that overexpression of p66shc leads to a decrease in Grb2 association with p52shc as well as decreased Grb2 membrane recruitment, strongly support these conclusions. Thus, the increase of Grb2 in the membrane fraction after IGF-I stimulation was greatly attenuated, and IGF-I-dependent MAPK activation was impaired by overexpression of p66shc.

Although all three isoforms of shc contain the important tyrosine phosphorylation sites, Tyr239/240 and Tyr317, previous studies have focused on p52shc and/or p46shc in mediating the mitogenic effect of growth factors, such as EGF, insulin (14,29,52), and IGF-I (7,12). Overexpression of p52/p46shc has been shown to enhance EGF-induced MAPK activation (4,14), whereas,overexpression of p66shc had no effect (4) or inhibited this response (14). In both studies, p66shc underwent tyrosine phosphorylation after EGF stimulation and formed a stable complex with Grb2. The authors proposed that Grb2-associated p66shc may interfere with the function of the recruited Grb2-SOS binding and consequently fail to enhance MAPK activation (4) or that overexpression p66shc competitively binds to a limited pool of Grb2, resulting in impaired p52shc association and MAPK activation (14). An inhibitory effect of p66shc on the MAPK activation was also observed in other cell types, including L6 cells (53), endothelial cells (54), and T cells (55). Interestingly, reduction of p66shc protein in L6 cells with antisense cDNA fragment was associated with increased basal Erk activation and blunted IGF-I-stimulated Erk activation (53), which is clearly different from the results in our current study. However, these previous studies did not address the question why Grb2 associated with tyrosine-phosphorylated p66shc did not lead to an increase in Ras/MAPK activation. Our findings extend these previous studies because the results show that a reduction in p66shc using RNAi leads to enhanced p52shc phosphorylation and Grb2 association. Furthermore, our results suggest that overexpression of p66shc reduces MAPK activation by disrupting SHPS-1/SHP-2/Src/p52shc/Grb2 complex formation because both p52shc phosphorylation and Grb2 binding to p52shc were inhibited. This results in decreased Grb2 membrane recruitment (as shown in Fig. 8), which contributes to decreased Erk activation. Recently, a poly-proline sequence located in a p66shc unique CH2 domain has been proposed to compete with Grb2 for binding to SOS (56). This could provide an alternative explanation for the inhibitory effect of p66shc on IGF-I-stimulated MAPK activation.

Vascular SMC proliferation and migration are key components of atherosclerotic lesion formation. Our previous studies have shown that IGF-I is a potent stimulator of SMC proliferation and migration via activation of PI3K/Akt and MAPK pathways (34,57) and disruption of IGF-I signal transduction impairs IGF-I-dependent cell proliferation and migration (12,13,58,59). Shc proteins, especially p52shc, have been shown to play a critical role in mediating IGF-I signal transduction, IGF-I-induced cell proliferation (7,9,10,11,12,13) and growth factor-induced cell migration (25,60,61). Therefore, disruption of shc signaling also affects cell proliferation and migration. Previous studies have shown that inhibition of the p52shc/Grb2 signaling pathways blocks angiotensin-dependent vascular SMC proliferation (62) and suppresses growth of tumors xenografted in nude mice (63). Our current study shows that p66shc negatively regulates IGF-I-dependent cell proliferation and migration in pSMC via impairment of IGF-I-induced MAPK. P66shc has been previously shown to act as a negative regulator of human and mouse T-cell survival and proliferation (55). However, results in human carcinoma cells correlating p66shc and cell proliferation are conflicting. Positive correlations were found in ovarian cancer cell lines (64), breast cancer cell lines (65), and prostate cancer cells (66), whereas other studies showed that p66shc protein level inversely correlated with the expression of ErbB-2, a prognostic marker for breast cancer cell lines (64), and the relapse of breast cancer (67). Therefore, in transformed cells, the effect of p66shc on cell proliferation appears to be cell type dependent.

In summary, these results clearly show that p66shc negatively regulates IGF-I signal transduction by inhibiting IGF-I-dependent p52shc tyrosine phosphorylation, Grb2 association with p52shc, and IGF-I stimulation of MAPK. Furthermore, in pSMC, p66shc acts as a negative regulator of IGF-I-dependent cell proliferation and migration. The results indicate that p66shc inhibits IGF-I signaling via inhibiting the SHPS-1/SHP-2/Src/p52shc/Grb2 complex formation after IGF-I stimulation, leading to impairment of Grb2 membrane recruitment and downstream signaling. These data provide the novel information for understanding how different isoforms of Shc regulate tyrosine kinase receptor-linked signaling pathways.

MATERIALS AND METHODS

Human IGF-I was a gift from Genentech (South San Francisco, CA). Polyvinyl difluoride membranes (Immobilon P) were purchased from Millipore Corp. (Billerica, MA). Autoradiographic film and ECL reagents were obtained from Pierce (Rockford, IL). DMEM containing 4500 mg glucose/liter (DMEM-H), penicillin, streptomycin, and blasticidin were purchased from Invitrogen (Carlsbad, CA). The Grb2, IGF-IR β-subunit, and 14-3-3β polyclonal antibodies and the monoclonal phosphotyrosine antibody (PY99) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SHPS-1 and SHP-2 antibodies were purchased from Millipore Corp. (Billerica, MA). The Src antibody was purchased from EMD Chemicals, Inc. (San Diego, CA). The shc antibodies, including the polyclonal and monoclonal, the Grb2 monoclonal antibody, and the caveolin antibody were purchased from BD Bioscience (San Diego, CA). The total Erk1/2, phospho-Erk1/2, and HA antibodies were obtained from Cell Signaling (Danvers, MA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Cell Culture

The pSMC were isolated from porcine aortas using a method that had been previously described (68). The cells were maintained in DMEM-H supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), streptomycin (100 μg/ml) and penicillin (100 U/ml). The cells that were used in these experiments were used between passages 5 and 15.

Construction of a Plasmid Containing Short Hairpin RNA (shRNA) Template for p66shc Silencing

Based on Invitrogen web site design tools, a sequence containing 19 oligonucleotides (GAATGAGTCTCTGTCATCG) located within the p66shc-specific CH2 domain (GenBank accession number NM_183001; nucleotides 42–60) was used to construct the shRNA template plasmid. The selected sequences were not found in any other mammalian protein searched using the NCBI Blast search program. The oligonucleotides were synthesized by Nucleic Acids Core Facility at University of North Carolina, annealed and ligated into BLOCK-iT U6 RNAi Entry Vector (catalog no. K4945-00; Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The complete sequence was verified by DNA sequencing. The expression vector was generated using the Gateway LR recombination reaction between the Entry Vector and BLOCK-iT Lentiviral RNAi Gateway Vector (catalog item K4943-00; Invitrogen). The empty vector (EV) was used as a control. After confirmation of the sequence, plasmid DNA was prepared by a Plasmid Mini Kit (Promega, Madison, WI).

Generation of pLenti-HA p66shc and pLenti-LacZ Vectors

Full-length human p66shc cDNA was generated by RT-PCR from mRNA that had been derived from human liver total RNA (Ambion, Austin, TX) and cloned into the pcDNA3.1V5H TA TOPO vector (Invitrogen). The full-length p66shc sequence was PCR amplified and cloned into the expression vector pLenti6/V5-D-TOPO (Invitrogen). The forward and reverse primers that were used to generate the PCR product were 5′-caccatgaatctcctgccccccaagcccaa-3′ and 5′-ttaAGCGTAATCTGGAACATCGTATGGGTAcagtttccgctccacaggttgctgta-3′. The PCR product containing a Kozak sequence (CACC) at the 5′ end of the p66shc coding sequence and a HA (uppercase letters) sequence at the 3′ end was cloned into the pLenti6/V5-D-TOPO expression vector. The complete sequences were verified by DNA sequencing. The pLenti-lacZ vector was generated using the Gateway LR recombination reaction between pLenti6/V5-GW/lacZ (catalog no. V496-10) and pLenti6/V5-D-TOPO expression vector (Invitrogen).

Generation of Virus Stocks

293FT cells (Invitrogen) were prepared for generation of virus stocks of each individual pLenti-construct. Cells were plated at 5 × 106 per 75-cm2 plate (Corning Glassworks, Corning, NY) the day before transfection in the growth medium (DMEM-H supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 U/ml penicillin). On the day of transfection, the culture medium was replaced with 5 ml Opti-MEM-I (Invitrogen) without antibiotics or serum. The DNA-Lipofectamine 2000 complexes that were used for each transfection were prepared according to the manufacturer’s protocol (Invitrogen), and 1.5 ml was added to the medium. The next day, the medium containing the DNA-Lipofectamine 2000 complexes was removed and replaced with the growth medium. The virus-containing supernatants were harvested 48–72 h after transfection and centrifuged at 3000 rpm for 15 min at 4 C to pellet the cell debris. The supernatants were filtered and stored as 1-ml aliquots at −80 C.

Establishment of pSMCs Expressing pLenti-p66shc shRNA, pLenti-HA p66shc, pLenti-EV, and pLenti-LacZ

pSMCs (passages 4 and 5) were plated at 3 × 105 per well in a six-well plate (353046, Falcon; BD Biosciences Discovery Labware, Bedford, MA) the day before transduction. The viral stocks were thawed, and the viral complexes precipitated as follows. For each 1 ml virus stock, 1 μl of an 80 mg/ml solution of chondroitin sulfate (C4384; Sigma-Aldrich) was added and then mixed gently and incubated at 37 C for 10 min. One microliter of 80 mg/ml polybrene (H9286; Sigma-Aldrich) was added and incubated at 37 C for 10 min. The mixture was centrifuged at 10,000 rpm for 5 min to pellet the virus, and the supernatant was removed. For transduction, the pellet was resuspended in 1 ml growth medium, and 1 μl of a 40 mg/ml solution of polybrene was added. The mixture was incubated with the cells for 24 h. The virus-containing medium was removed and changed to 2 ml growth medium for an additional 24 h and then replaced with selection medium (growth medium containing 4 μg/ml blasticidin; Invitrogen, San Diego, CA). The cultures were then grown to confluence. The expression of HA-tagged p66shc protein was detected by immunoblotting with a 1:1000 dilution of anti-HA antibody.

Immunoprecipitation and Immunoblotting

Cells were seeded at 5 × 105 cells per 10-cm plate (BD Biosciences, Franklin Lakes, NJ) and grown for 6 d to reach confluence. The cultures were incubated in serum-free DMEM-H for 16–18 h before the addition of IGF-I (100 ng/ml). The cell monolayers were lysed in a modified radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40; 0.25% sodium deoxycholate; 1 mm EGTA; 150 mm NaCl; and 50 mm Tris-HCl, pH 7.5) in the presence of protease inhibitors (10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml pepstatin), and phosphatase inhibitors (25 mm sodium fluoride and 2 mm sodium orthovanadate). The cell lysates were centrifuged at 14,000 × g for 10 min at 4 C. The supernatants containing crude membrane and cytosolic proteins were exposed to the following dilutions of anti-shc (1:300), anti-PY99 (1:200), anti-Grb2 (1:200), IGF-IR (1:300), anti-SHPS-1 (1:500), anti-Src (1:500), or anti-SHP-2 (1:500) antibody overnight at 4 C. The immunoprecipitates were immobilized using protein A beads for 2 h at 4 C and washed three times with the same lysis buffer containing protease and phosphatase inhibitors. The precipitated proteins were eluted in 40 μl 2× Laemmli sample buffer, boiled for 5 min, and separated using 10% SDS-PAGE. The proteins were transferred to Immobilon-P membranes that were blocked for 1 h in 3% BSA or 5% nonfat milk in Tris-saline buffer with 0.2% Tween 20. The blots were incubated overnight at 4 C with the indicated antibodies (1:1000 for anti p-Tyr, SHPS-1, IGF-IR, Src, SHP-2, caveolin, 14-3-3β, or shc and 1:5000 for anti-Grb2). To detect Erk1/2 activation, 20 μl cell lysate was removed before immunoprecipitation and mixed with 20 μl 2× Laemmli sample buffer and then separated by 10% SDS-PAGE. An anti-phospho-Erk1/2 (1:1000) antibody was used to detect phosphorylated Erk1/2, and total Erk1/2 was detected using a polyclonal anti-Erk1/2 antibody (1:1000). The proteins were visualized using enhanced chemiluminescence (Pierce).

Isolation of Cytoplasmic and Membrane Fraction Proteins

The pLenti-LacZ and pLenti-HA p66shc cells were grown to confluence and were serum starved 16–18 h before IGF-I treatment. After IGF-I stimulation for the indicated times, two 10-cm plates were lysed with 1.5 ml ice-cold PBS containing protease and phosphatase inhibitors and transferred to a new tube. The lysates were frozen at −80 C for 1 h and thawed at room temperature. After three cycles, they were centrifuged at 13,000 × g for 25 min. The supernatant was transferred to a new tube and labeled as the cytoplasmic fraction. The pellet was suspended with 0.75 ml membrane protein isolation buffer (20 mm Tris-HCl; 150 mm NaCl; 1 mm EDTA; 1 mm EGTA; and 1% Triton X-100, pH 7.5) containing protease and phosphatase inhibitors. The lysates were treated with ultrasonication briefly and centrifuged at 13,000 × g for 25 min. The supernatant was transferred to another new tube and considered as the solubilized membrane fraction. Caveolin and 14-3-3β proteins were used as markers for the membrane and cytoplasmic fractions, respectively.

Cell Proliferation Assay

Assessment of pSMC proliferation was performed as described previously (69). Cells were incubated with or without IGF-I (50 ng/ml) in serum-free DMEM-H containing 0.2% platelet-poor plasma for 48 h, and cell number in each well was counted. Each treatment was analyzed in triplicate, and the results represent mean values of three independent experiments.

Cell Migration Assay

Wounding of control, p66shc knockdown and overexpression pSMC were performed as previously description (25). The wounded monolayers were exposed to IGF-I (100 ng/ml) for 48 h at 37 C. The cells were fixed and stained (Diff-Quick; Dade Behring, Inc., Newark, DE), and the number of cells migrating into the wound area was counted. At least eight of the previously selected 1-mm areas at the edge of the wound were counted for each data point.

Statistical Analysis

Chemiluminescent images obtained were scanned using a DuoScan T1200 (AGFA, Brussels, Belgium), and band intensities of the scanned images were analyzed using National Institutes of Health Image J program, version 1.37. To correct for gel loading differences and differences in the efficiency of immunoprecipitation, the results are expressed as the scanning units divided by the scanning units obtained when the amount of protein that was immunoprecipitated was quantified by immunoblotting. The results that are shown in all experiments are the representative of at least three separate experiments and expressed as the mean ± sem of three independent experiments. The Student’s t test was used to compare differences between treatments.

Acknowledgments

We thank Dr. Jane Badley-Clarke for her help in vector construction and Ms. Laura Lindsey for her help in preparing the manuscript.

Footnotes

This work was funded by National Institutes of Health Grant AG-02331 to D.R.C.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 7, 2008

Abbreviations: EGF, Epidermal growth factor; EVC, empty vector control; Grb2, growth factor receptor-bound protein-2; HA, hemagglutinin; IGF-IR, IGF-I receptor; pSMC, porcine SMC; RNAi, RNA interference; Shc, Src homology collagen; SHP-2, Src homology 2 domain-containing protein tyrosine phosphatase-2; SHPS-1, SHP substrate-1; shRNA, short hairpin RNA; SMC, smooth muscle cells; SOS, son of sevenless.

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