Tyrosine Phosphorylation of the ß3-Subunit of the {alpha}Vß3 Integrin Is Required for Membrane Association of the Tyrosine Phosphatase SHP-2 and Its Further Recruitment to the Insulin-Like Growth Factor I Receptor

Yan Ling, Laura A. Maile and David R. Clemmons

University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, M.D., CB-7170, 6111 Thurston-Bowles, Division of Endocrinology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7170. E-mail: endo{at}med.unc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand occupancy of the {alpha}Vß3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the {alpha}Vß3 integrin on ß3 phosphorylation and studied the role of ß3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to {alpha}Vß3 induced tyrosine phosphorylation of the ß3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with ß3. In confluent SMCs, the ß3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in ß3 phosphorylation and resulted in decreased SHP-2 association with ß3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant ß3 that had two tyrosines changed to phenylalanines showed a 89.9 ± 1.2% decrease in ß3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated ß3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant ß3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of {alpha}Vß3 regulates the SMC response to IGF-I.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INTEGRIN RECEPTORS ARE heterodimeric membrane proteins that link extracellular matrix components with intracellular signaling events (1). Our previous studies have shown that ligand occupancy of the {alpha}Vß3 integrin is required for IGF-I stimulation of smooth muscle cell (SMC) migration and DNA synthesis (2, 3). We have further demonstrated that activation of IGF-I receptor (IGF-IR)-linked downstream signaling events, such as activation of the phosphatidylinositol 3 kinase (PI-3 kinase) and MAPK pathways by IGF-I, is dependent on {alpha}Vß3 occupancy. These findings suggest that this integrin receptor has a significant role in regulating IGF-IR signaling (3, 4).

Integrin receptors have been shown to regulate growth factor signaling through the protein tyrosine phosphatase SHP-2 (5). SHP-2 is a nontransmembrane tyrosine phosphatase that has been shown to bind to and dephosphorylate the GH receptor, the platelet derived growth factor receptor (PDGFR), and IGF-IR (5, 6, 7, 8). Integrin receptor occupancy has been shown to increase the recruitment of SHP-2 to the PDGFR (5). The enhanced association of SHP-2 with PDGFR decreases duration of receptor phosphorylation, thereby decreasing receptor-mediated activation of the GTPase-activating protein of Ras. This leads to enhanced PDGFR-dependent cellular responses (5). In primary cultured vascular smooth muscle cells (pSMCs), SHP-2 is recruited to IGF-IR in response to IGF-I stimulation and it subsequently dephosphorylates the receptor (6). The regulation of SHP-2 recruitment to the IGF-IR is therefore a key step in the control of IGF-I-dependent signaling and has the potential to be regulated by integrin occupancy as is the case for the PDGFR.

In addition to the regulatory role of SHP-2 via its binding to growth factor receptors, several studies have also shown that proper recruitment of SHP-2 to the signaling molecule complexes is required for downstream signaling after growth factor receptor activation (9, 10). Our previous studies have demonstrated that IGF-I stimulation results in tyrosine phosphorylation of the transmembrane protein SHPS-1 (Src homology 2 domain-containing tyrosine phosphatase substrate 1), which leads to recruitment of SHP-2 to SHPS-1 and subsequent activation of SHP-2 phosphatase activity (6). The association of SHP-2 with SHPS-1 is a prerequisite for SHP-2 recruitment to the IGF-IR since expression of a truncated form of SHPS-1 that did not bind SHP-2 resulted in impaired recruitment to IGF-IR. After SHP-2 binding to SHPS-1, activated SHP-2 dephosphorylates SHPS-1, resulting in its disassociation, and subsequently it is recruited to activated IGF-IR (6). The catalytic activity of SHP-2 is also required for downstream IGF-I signaling since overexpression of a catalytically inactive SHP-2 results in inhibition of MAPK activation and cell migration in response to IGF-I (11). Inhibition of SHP-2 binding to SHPS-1 also leads to impaired IGF-I-stimulated SMC migration (12). Similarly, since blocking ligand occupancy of {alpha}Vß3 also inhibits IGF-I-stimulated cell migration and DNA synthesis, {alpha}Vß3 may play a role in regulating SHP-2 transfer and activation.

In platelets after ligand occupancy and integrin clustering, the cytoplasmic domain of the ß3-subunit can undergo phosphorylation on its two-tyrosine residues (Y747 and Y759) (13). The phosphorylation of the ß3-subunit has been shown to be responsible for triggering downstream signaling events, including activation of the MAPK pathways (14, 15). Furthermore, blocking ß3 phosphorylation by mutating Y747 and Y759 to phenylalanine has been shown to block IGF-I-stimulated cell migration (4). Whereas a role for ß3 phosphorylation in IGF-I signaling is apparent, it is not known how the tyrosine phosphorylation status of the ß3-subunit regulates IGF-IR-linked signaling. In the current study, we analyzed the role of ligand occupancy of the {alpha}Vß3 integrin in controlling ß3 phosphorylation. Because SHP-2 is a key regulator of IGF-I signaling, we determined the impact of altering ß3-subunit phosphorylation on the regulation of SHP-2 recruitment to other signaling molecules and on IGF-I-dependent biological responses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand Occupancy of the {alpha}Vß3 Induces ß3 Phosphorylation and SHP-2 Association
To determine the effects of ligand occupancy of the {alpha}Vß3 integrin on IGF-IR signaling, we first determined the effects of adding vitronectin on {alpha}Vß3 phosphorylation. In subconfluent SMCs, there was no detectable phosphorylation of the ß3-subunit. In contrast, incubation of cells with vitronectin induced ß3 phosphorylation (P < 0.05) while the ß3 protein level was not changed (Fig. 1Go). When SHP-2 coimmunoprecipitation with the ß3-subunit was analyzed, SHP-2 binding to ß3 was detected only in subconfluent cells that had been exposed to vitronectin. When cells were grown to confluency, there was constitutive phosphorylation of the ß3-subunit, and SHP-2 was also constitutively associated with ß3. Addition of exogenous vitronectin to the confluent cultures further enhanced tyrosine phosphorylation of the ß3-subunit (P < 0.05) with a corresponding increase of SHP-2 association (P < 0.05). These results show that tyrosine phosphorylation of ß3 is regulating its association with SHP-2.



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Fig. 1. Ligand Occupancy-Induced ß3 Phosphorylation and SHP-2 Association

Subconfluent and confluent cultures were serum starved overnight and incubated with or without vitronectin (2 µg/ml) for 2 h. Cells were then lysed in the Triton X-100 containing buffer, and equal amounts of total protein were immunoprecipitated with anti-ß3 antiserum described in Materials and Methods. ß3 phosphorylation and SHP-2 association were determined by immunoblotting with an antiphosphotyrosine (p-Tyr) (upper panel) or anti-SHP-2 antibody (middle panel). ß3 protein level was evaluated by immunoblotting with anti-ß3 antibody (lower panel). The immunoprecipitates were quantified using scanning densitometry. The results are expressed as arbitrary units and are shown as the mean ± SEM (n = 3). *, P < 0.05 when treatment with vitronectin is compared with control non-vitronectin-treated cultures.

 
Inhibition of ß3-Subunit Phosphorylation Blocks SHP-2 Recruitment to ß3 and the Cell Membrane
To confirm that the association between ß3 and SHP-2 is tyrosine phosphorylation dependent, we incubated wild-type pSMCs with the Src kinase inhibitor PP2 since it has been shown that a Src family kinase is responsible for inducing ß3 phosphorylation (16). In confluent SMCs, ß3 was constitutively phosphorylated, whereas incubation of confluent cultures with PP2 showed a concentration-dependent inhibition on ß3 phosphorylation (Fig. 2Go, A and D). Correspondingly, we detected a parallel decrease in SHP-2 association with the ß3-subunit. To control for nonspecific kinase inhibition, control cultures were exposed to the same concentration of PP2, and IGF-IR phosphorylation was determined. PP2 did not inhibit IGF-I-stimulated activation of the IGF-IR kinase (Fig. 2BGo). Because SHP-2 is a cytosolic phosphatase, we hypothesized that by associating with phosphorylated ß3, SHP-2 may be recruited to the vicinity of the cell membrane therefore retained in the membrane-associated fraction. Figure 2Go, C and D, shows that after exposure to increasing concentrations of PP2, the amount of SHP-2 localized to the Triton X-100 soluble membrane fraction was decreased. In contrast, we detected increasing amounts of SHP-2 in the cytosolic fractions while the total amount of SHP-2 was not changed. These results provide direct evidence that tyrosine phosphorylation of the ß3-subunit plays a key role in recruiting SHP-2 to the cell membrane via association with ß3, and inhibition of ß3 phosphorylation abolishes SHP-2 membrane association.



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Fig. 2. Concentration-Dependent Effects of PP2 on ß3 Phosphorylation, SHP-2 Association with ß3, and SHP-2 Membrane Association

Confluent cultures were serum starved overnight in the presence or absence of the Src family kinase inhibitor PP2 at various concentrations. Cells were lysed in the Triton X-100-containing buffer, and an equal amount of total protein was immunoprecipitated with anti-ß3 or anti-SHP-2 antibody to evaluate total SHP-2 protein content. Membrane-associated SHP-2 was evaluated by immunoprecipitation and immunoblotting of SHP-2 from membrane extracts. Cytosolic SHP-2 was detected by immunoblotting with anti-SHP-2 antibody using 30 µg of total protein from cytosolic extracts. A, Representative immunoblots of phospho-ß3 (upper panel), SHP-2 association (middle panel), and total ß3 protein (lower panel). B, Confluent cultures were treated with PP2 (4 µM) overnight and then stimulated with IGF-I for 5 min. The cell lysates were immunoprecipitated with the anti-IGF-IR and immunoblotted with anti p-Tyr or anti-IGF-IR. C, Representative immunoblots of membrane-associated SHP-2 (upper panel), cytosolic SHP-2 (middle panel), and total SHP-2 protein (lower panel). D, The results were quantified using scanning densitometry and plotted as arbitrary units. Each point is the mean ± SEM of three separate experiments.

 
Impaired SHP-2 Recruitment to {alpha}Vß3 in SMCs Expressing the Nonphosphorylated ß3-Subunit
To understand the significance of ß3 phosphorylation in regulating SHP-2 recruitment and therefore IGF-IR signaling, we established SMCs expressing a mutant form of ß3 in which tyrosine 747 and 759 had been substituted with phenylalanine (ß3-FF). As illustrated in Fig. 3Go, cells transfected with mutant pMEP4-ß3-FF/FLAG construct express the FLAG-labeled ß3 protein while the vector-transfected cells have no detectable FLAG ß3 protein (Fig. 3AGo). The mutant ß3-FF protein is expressed in the transfected cells at a level that is similar to the level of expression of wild-type, endogenous ß3-subunit (ß3-WT) in the vector-transfected cells (Fig. 3BGo). In the basal state, when pSMCs are confluent and stably attached, the ß3-WT subunit is tyrosine phosphorylated while cells expressing mutant ß3-FF show minimal phosphorylation (Fig. 3CGo) that is equivalent to 11.1 ± 1.2% (mean ± SEM; n = 3) of that in control cells. This low level of phosphorylated ß3 in the ß3-FF cells is likely due to the residual endogenous ß3. Thus, expression of ß3-FF acts in a dominant negative manner and greatly reduces the total amount of phosphorylated ß3. This level of FLAG ß3 expression is representative of levels that were present in the subsequent experiments.



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Fig. 3. Establishment of pSMCs Expressing Mutant ß3-FF

The partial sequence of the cytoplasmic domain of the ß3-subunit is shown indicating the location of the two tyrosine residues that were changed to phenylalanines to generate the ß3-FF mutant. Cells transfected with either the pMEP4 vector (vector) or pMEP4-ß3-FF/FLAG (ß3-FF) were grown to confluency and lysed in buffer containing 1% Triton X-100. A, Cell lysates were immunoprecipitated with M1 anti-FLAG monoclonal antibody and immunoblotted with a polyclonal anti-ß3 antibody. B, The cell lysates were immunoprecipitated and immunoblotted with a polyclonal anti-ß3 antibody. C, Cell lysates were immunoprecipitated with a polyclonal anti-ß3 antibody and immunoblotted with antiphosphotyrosine (p-Tyr).

 
SHP-2 Association with the ß3-Subunit and Association with the Cell Membrane Is Disrupted in ß3-FF Cells
Using coimmunoprecipitation and immunoblotting, we found that SHP-2 is associated with the ß3-WT subunit but not with the mutant ß3-FF in confluent cultures (Fig. 4AGo). To exclude the possibility that exogenously expressed ß3 may interfere with SHP-2 binding, we analyzed the association of SHP-2 with ß3 in cells that were transfected with a ß3-WT construct and compared it to cells transfected with ß3-FF. The result confirmed that SHP-2 was coimmunoprecipitated with the ß3-WT subunit but not the ß3-FF subunit (Fig. 4BGo). Correspondingly, SHP-2 membrane association was also disrupted in ß3-FF cells compared with vector-transfected cells (Fig. 4CGo). In vector-transfected cells, 49.5 ± 2.2% of the total SHP-2 (mean ± SEM, n = 3) is located in the membrane fraction while in cells expressing ß3-FF, only 4.8 ± 0.2% of the total SHP-2 (n = 3) was detected in the membrane-associated fraction. Importantly, there was no significant difference in total amount of SHP-2 between these two types of cells. The change in amount of SHP-2 in the membrane-associated fraction is specific because the amount of IGF-IR extracted in this fraction is not different between vector-transfected and ß3-FF cells (Fig. 4CGo). These results confirm that phosphorylation of the ß3-subunit is required for the localization of SHP-2 to the membrane fraction.



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Fig. 4. SHP-2 Association with ß3 and Its Distribution Between Cytosol and the Membrane-Associated Fraction in Vector-Transfected Cells and ß3-FF Cells

A, Vector-transfected cells and ß3-FF cells were serum starved overnight. The cells were then lysed in buffer containing Triton X-100 and immunoprecipitated with a polyclonal anti-ß3 antibody and subsequently immunoblotted with an anti-SHP-2 antibody (upper panel). The experiments were repeated three times, and similar results were obtained. To confirm that there were no loading differences, the blot was reprobed with an anti-ß3 antibody (lower panel). B, Upper panel: Cells transfected with ß3-WT construct or ß3-FF were serum depleted overnight and immunoprecipitated with the anti-ß3 antibody and immunoblotted with SHP-2 antibody. Lower panel: In a separate experiment, cell surface proteins from wild type ß3 or ß3-FF transfected cells were biotinylated. Cells were lysed and subjected to immunoprecipitation with antihuman ß3 antibody and subsequently blotted with antiavidin antibody. C, Confluent cultures were serum starved overnight and washed with PBS twice and then dissociated with 2 ml of cell dissociation buffer (see Materials and Methods). Cytosolic and membrane-associated proteins were separated after which immunoprecipitation was carried out using an antibody for SHP-2 followed by immunoblotting with the same anti-SHP-2 antibody. The experiment was repeated three times and similar results were obtained. As control, total SHP-2 protein contained in the lysates of vector-transfected cells and ß3-FF cells is shown after immunoprecipitation and immunoblotting with the anti-SHP-2 antibody. The distribution of IGF-IR in vector-transfected cells and ß3-FF cells is also shown after immunoprecipitation and immunoblotting with the anti-IGF-IR antibody.

 
Inhibition of SHP-2 Transfer to ß3-Subunit Alters Its Transfer to IGF-IR and SHPS-1
To determine the consequences of impaired SHP-2 membrane association, we first compared recruitment of SHP-2 to IGF-IR in cells expressing the ß3-FF mutant and vector-transfected cells. Figure 5AGo shows that, in cells expressing endogenous ß3 (vector transfected) after IGF-I stimulation, there is a rapid increase in IGF-IR phosphorylation followed by dephosphorylation after 20 min. At this time point, IGF-IR phosphorylation is decreased by 56.1 ± 7.7% (mean ± SEM, n = 3) compared with the level of phosphorylation after 5 min of IGF-I stimulation. This is consistent with our previous report that showed that SHP-2 binding to IGF-IR is detected after 20 min, coinciding with the decrease in receptor phosphorylation (6). In contrast, in the ß3-FF cells, whereas the increase in IGF-IR phosphorylation at 5 min is similar to the vector-transfected cells, there is no significant decrease in receptor phosphorylation by 20 min (3.4 ± 7.0% decrease compared with the band intensity detected after 5 min of IGF-I stimulation, n = 3). In addition, there is no detectable association between SHP-2 and the IGF-IR. The sustained IGF-IR phosphorylation and loss of SHP-2 transfer to the IGF-IR in ß3-FF cells is not due to expression of exogenous ß3. This is confirmed by the fact that cells transfected with ß3-WT show a similar decrease of IGF-IR phosphorylation after 20 min of exposure to IGF-I and SHP-2 is recruited to the IGF-IR at 20 min after IGF-I stimulation (Fig. 5BGo). These results demonstrate that failure to phosphorylate ß3 is associated with loss of SHP-2 transfer to IGF-IR.



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Fig. 5. Lack of Association between SHP-2 and the IGF-IR in Cells Expressing Mutant ß3-FF Protein

A, pMEP4 vector-transfected cells (vector) and pMEP4-ß3-FF/FLAG-transfected cells (ß3-FF) were serum starved overnight and exposed to IGF-I (100 ng/ml) for 5, 10, and 20 min. Cells were lysed in modified RIPA buffer and immunoprecipitated with an anti-IGF-IR antibody. Receptor phosphorylation and SHP-2 association were then determined by immunoblotting with either an antiphosphotyrosine (p-Tyr) (upper panel) or anti-SHP-2 antibody (middle panel). To control for receptor levels the blot was then reprobed with an anti-IGF-IR polyclonal antibody (lower panel). The results were quantified using scanning densitometry. The values for IGF-IR phosphorylated band intensities have been adjusted for IGF-IR protein levels. The results are plotted as a percentage of the maximum value obtained (5 min after IGF-I treatment). Each point is the mean ± SEM of three separate experiments. B, pMEP4-ß3-WT-transfected cells (ß3-WT) were serum depleted overnight and exposed to IGF-I (100 ng/ml) for 5, 10, or 20 min. The cells were lysed in the modified RIPA buffer and immunoprecipitated with the anti-IGF-IR antibody and subsequently immunoblotted with anti-p-Tyr and SHP-2 antibodies.

 
Effects of Nonphosphorylated ß3-Integrin on SHP-2 Association with Its Substrate SHPS-1
Since we have previously shown that transfer of SHP-2 to IGF-IR requires phosphorylation of SHPS-1 and SHP-2 transfer to SHPS-1 after its phosphorylation, we analyzed these events after IGF-I stimulation in the ß3-FF and vector-transfected cells. Figure 6Go shows that in vector-transfected cells, consistent with our previous results (6), there is a marked increase in SHPS-1 phosphorylation after 5 min of IGF-I stimulation (5.15 ± 1.48-fold increase, n = 4). This is accompanied by an increase in SHP-2 association followed by rapid SHPS-1 dephosphorylation and SHP-2 disassociation from SHPS-1. In contrast, there is no association between SHP-2 and SHPS-1 in ß3-FF cells either basally or after IGF-I stimulation. This failure of SHP-2 to bind to SHPS-1 is not due to loss of SHPS-1 phosphorylation since a 5.34 ± 1.03-fold increase (n = 4) in SHPS-1 tyrosine phosphorylation can still be detected 5 min after IGF-I. However, in contrast to cells expressing ß3-WT, there is no dephosphorylation of SHPS-1, which is consistent with the lack of SHP-2 association (Fig. 6Go). Taken together, these results indicate that, in cells expressing ß3-FF, although IGF-I activation of the IGF-IR is associated with SHPS-1 phosphorylation, SHP-2 transfer to SHPS-1 does not occur. This suggests that tyrosine phosphorylation of the ß3-subunit is required for SHP-2 recruitment to SHPS-1 after IGF-I stimulation.



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Fig. 6. SHP-2 Is Not Associated with Its Substrate SHPS-1 in Cells Expressing ß3-FF Protein

pMEP4 vector-transfected cells (vector) and pMEP4-ß3-FF/FLAG (ß3-FF)-transfected cells were serum starved overnight and then exposed to IGF-I (100 ng/ml) for 5, 10, and 20 min. Cells were then lysed in the modified RIPA buffer and immunoprecipitated with a polyclonal antibody against SHPS-1 and then subsequently immunoblotted with an antiphosphotyrosine (p-Tyr) (upper panel) or anti-SHP-2 antibody (middle panel). SHPS-1 protein levels are shown in the lower panel as indicated by immunoblotting with anti-SHPS-1 antibody. The results were quantified using scanning densitometry. The values for phosphorylated SHPS-1 have been adjusted to convey the differences in SHPS-1 protein levels. The results are plotted as a percentage of the maximum value obtained (5 min after IGF-I treatment). Each point is the mean ± SEM of four separate experiments.

 
IGF-I-Dependent DNA Synthesis Is Impaired in Cells Expressing ß3-FF Mutant
To determine functional consequence of impaired SHP-2 recruitment induced by lack of ß3 phosphorylation, we determined the ability of IGF-I to stimulate [3H]thymidine incorporation in nontransfected SMCs and in cells expressing ß3-FF. Figure 7Go shows that in control cells, IGF-I (50 ng/ml) induces a 4.5 ± 0.42 (n = 3)-fold increase of thymidine incorporation. However, the response to IGF-I is significantly impaired in cells expressing ß3-FF (e.g. 2.0 ± 0.33-fold increase), a 55.8 ± 4.2% (P < 0.01) reduction compared with the response of control cells. These results indicate that inhibition of ß3 phosphorylation impairs IGF-I-dependent mitogenic actions, consistent with our previous findings that ligand occupancy of the {alpha}Vß3 integrin is required for IGF-I-induced cellular migration and DNA synthesis (2, 3).



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Fig. 7. Impaired IGF-I-Dependent DNA Synthesis in Cells Expressing Mutant ß3-FF

The change in [3H]thymidine incorporation into DNA after IGF-I treatment was measured in nontransfected pSMCs and cells expressing mutant ß3-FF (pSMC-ß3FF). The results were plotted as fold increase of basal value. Each point is the mean ± SEM (n = 3 per experiment) and include the results from three separate experiments. *, P < 0.01 when treatment with 50 ng/ml IGF-I in control cells was compared with 50 ng/ml IGF-I in pSMC-ß3-FF.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our previous studies have shown that ligand occupancy of the {alpha}Vß3 integrin is required for SMCs to migrate in response to IGF-I and is necessary for full activation of the PI-3 kinase and MAPK pathways after IGF-IR activation (2, 3). Furthermore, we and others have shown that recruitment of catalytically activated SHP-2 is essential for the appropriate duration of IGF-IR phosphorylation and cellular responses to activated IGF-IR (4, 6, 11). Therefore, to determine the mechanism by which ligand occupancy of the {alpha}Vß3 integrin was altering IGF-IR signaling, we investigated the role of {alpha}Vß3 integrin activation that is induced by ligand occupancy in regulating SHP-2 membrane recruitment and transfer to SHPS-1 and IGF-IR. Our results show that tyrosine phosphorylation of the ß3-subunit of the {alpha}Vß3 integrin is an important regulator of SHP-2 recruitment to ß3. Since ligand occupancy stimulates ß3 phosphorylation, these studies provide a link between {alpha}Vß3 ligand occupancy and SHP-2 recruitment to both the ß3-subunit and to the cellular membrane fraction.

Several previous studies that used stably attached cells have shown that ligand binding to growth factor receptors leads to phosphorylation of SHPS-1 and to SHP-2 recruitment (17, 18, 19, 20, 21) while failure to phosphorylate SHPS-1 blocks the SHP-2 recruitment. These studies concluded that phosphorylation of SHPS-1 resulted in recruitment of SHP-2 from the cytosol to the membrane fraction and that this was necessary for the positive regulatory effects of SHP-2 on growth factor-dependent downstream signaling (17, 19). However, none of those studies determined the role of integrin receptor ligand occupancy or ß3-subunit phosphorylation in SHP-2 membrane localization or its recruitment to SHPS-1, and in one study analysis of the effect of SHPS-1 phosphorylation on regulating SHP-2 membrane recruitment was carried out in Chinese hamster ovary cells that do not express {alpha}Vß3 integrin (22). Our current studies extend those observations by showing that although IGF-I induced SHPS-1 phosphorylation is required for SHP-2 transfer to IGF-IR, SHPS-1 phosphorylation alone is not sufficient. Our results show that in cells expressing ß3-FF, the basal association of SHP-2 with the cell membrane and with the ß3-subunit is disrupted; therefore, SHP-2 is not recruited to SHPS-1 despite increased SHPS-1 phosphorylation after IGF-I stimulation. Since ligand occupancy of {alpha}Vß3 or confluent cell density result in substantial increases in ß3 phosphoryation and SHP-2 membrane localization, taken together these findings suggest that ligand occupancy of the {alpha}Vß3 integrin leads to the recruitment of SHP-2, thus allowing its transfer to membrane-associated signaling molecules such as SHPS-1 and IGF-IR after IGF-I stimulation (Fig. 8Go).



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Fig. 8. Schematic Diagram Describing the Role of ß3 Phosphorylation on SHP-2 Recruitment to the Cell Membrane (CM) and Its Further Transfer to SHPS-1 and to IGF-IR

Top panel, In confluent SMC cultures the ß3-subunit of {alpha}Vß3 is basally tyrosine phosphorylated, and SHP-2 associates with ß3 via an unknown intermediary protein. Ligand occupancy of the {alpha}Vß3 or increased cell density enhances ß3 phosphorylation, resulting in increased SHP-2 association. Inhibition of ß3 phosphorylation by either the addition of the tyrosine kinase inhibitor PP2 or mutation of the two ß3 tyrosines to phenyalanines (ß3-FF) abolishes SHP-2 binding to ß3 and the association of SHP-2 with the cell membrane. Lower panel, Upon IGF-I stimulation, SHPS-1 is tyrosine phosphorylated and the membrane-associated SHP-2 is recruited to SHPS-1. Subsequently, SHP-2 is activated and it dephosphorylates SHPS-1, resulting in SHP-2 disassociation and transfer to the activated IGF-IR. Inhibition of SHP-2 membrane association resulting from impaired ß3 phosphorylation attenuates SHP-2 recruitment to SHPS-1 in response to IGF-I, thus resulting in impairment of SHP-2 transfer to IGFIR.

 
After cellular reattachment, integrin engagement has been shown to be associated with phosphorylation of SHPS-1 and recruitment of SHP-2 (23). The formation of this SHP-2-SHPS-1 complex has been proposed to be important for regulating early events in integrin signaling (24). However, those observations were made using cells that had been recently detached and reattached to extracellular matrix-coated dishes. Although those experiments provide useful information of attachment-mediated integrin signaling, they do not predict the results that would be obtained using stably attached cells when analyzing how integrin engagement affects intracellular signaling of attached cells in response to growth factor stimulation. Our results extend those observations by demonstrating that in confluent, stably attached cells, the ß3-subunit is constitutively phosphorylated and that modulation of its phosphorylation status can regulate SHP-2 recruitment to the cell membrane.

The role of ß3 phosphorylation in the regulation of SHP-2 membrane localization was analyzed in two ways. Since a Src family kinase has been shown to mediate phosphorylation of the ß3-subunit (16), we used the Src family kinase inhibitor PP2 to inhibit ß3 phosphorylation. PP2 decreased SHP-2 association with ß3 and the amount of SHP-2 in the membrane-associated fraction, allowing us to conclude that phosphorylation of ß3 regulates SHP-2 association. This conclusion was further substantiated by showing that cells expressing a form of ß3 that cannot be phosphorylated had no SHP-2 recruitment to ß3-FF or to the cell membrane, whereas cells expressing an equivalent level of ß3-WT showed SHP-2 transfer to ß3 and IGF-IR. Because expression of ß3-FF decreased the level of endogenous phosphorylated ß3, these results suggest that failure to recruit SHP-2 to ß3 was a direct consequence of the reduced ß3 phosphorylation.

Whether SHP-2 binds directly to phosphorylated ß3 integrin or whether this interaction occurs via an intermediary protein was not addressed. We were unable to directly coprecipitate SHP-2 from cell membrane extracts when synthetic ß3 peptides that contained phosphorylated Tyr 747 and Tyr 759 residues were used in vitro, suggesting that there is no direct association between the C-terminal region of ß3 and SHP-2 (data not shown). This conclusion is supported by a recent study showing that SHP-2 does not bind to ß3 in vitro unless its amino acid sequence is altered (25). Therefore, it is likely that ligand-induced ß3 tyrosine phosphorylation allows an unidentified protein to localize SHP-2 to the membrane in a complex with the phosphorylated ß3-subunit. A possible candidate linker protein is the insulin receptor substrate 1 (IRS-1), a major signaling mediator that is activated by both the insulin receptor and IGF-IR. Vuori and Ruoslahti (26) showed that stimulation of insulin receptor in rat fibroblasts that overexpressed insulin receptors and a pancreatic tumor cell line that had been transfected with {alpha}V led to the binding of phosphorylated IRS-1 to {alpha}Vß3. In our system, however, we were unable to coprecipitate IRS-1 with the ß3-subunit either basally or upon IGF-I stimulation (data not shown). This difference could be due to the fact that we use primary cultured vascular SMCs that do not overexpress growth factor receptors or {alpha}Vß3.

Our previous studies have indicated that association of SHP-2 with IGF-IR provides a regulatory mechanism for controlling the duration of IGF-IR phosphorylation (6). The significance of SHP-2 transfer to growth factor receptors, particularly its impact on receptor downstream signaling, has been extensively studied. In some studies SHP-2 has been shown to be a negative regulator of growth factor receptor-mediated signaling (20, 21, 27). For example, increased recruitment of SHP-2 to SHPS-1 inhibits GH signaling (28), and blocking SHP-2 binding to GH receptor results in enhanced STAT5B activation (29). Inhibition of SHP-2 binding to IRS-1 was shown to lead to enhanced PI-3 kinase activation after insulin stimulation (30). In contrast, other studies have shown that increased transfer of SHP-2 to growth factor receptors results in enhancement of growth factor-stimulated actions (5, 7). Additional studies have demonstrated that transfer of catalytically activated SHP-2 to signaling proteins in both the PI-3 kinase and MAPK pathways is necessary for sustained pathway activation (31, 32, 33, 34, 35). Consistent with this concept, our previous studies have shown that the ß3-FF cells do not migrate in response to IGF-I (4), and the current results indicate that failure to transfer SHP-2 also impairs IGF-I-stimulated DNA synthesis. Because our prior studies have shown that the PI-3 kinase pathway activation is required for IGF-I-stimulated cell migration and MAPK activation is necessary for increased DNA synthesis (36), it is probable that impaired SHP-2 transfer will alter IGF-I receptor-linked signaling through both pathways. Since transfer of SHP-2 to downstream signaling complexes is necessary for pathway activation (9, 10) and for IGF-I stimulated biological actions (4, 11), our studies indicate a potential role for ß3 phosphorylation in the modulation of SHP-2 transfer to signaling molecules that are downstream from the IGF-I receptor and suggest that loss of SHP-2 transfer plays a role in {alpha}Vß3 antagonist-mediated inhibition of the cell migration and DNA synthesis responses to IGF-I.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials and Antibodies
Human IGF-I was a gift from Genentech (South San Francisco, CA). Immobilon-P membranes were purchased from Millipore Corp. (Bedford, MA). DMEM containing 4500 mg glucose/liter (DMEM-H) was purchased from Life Technologies (Gaithersburg, MD), and streptomycin and penicillin were purchased from Invitrogen (San Diego, CA). Polyclonal antibodies for SHP-2 and SHPS-1 were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). A polyclonal antibody for the ß3-subunit of porcine {alpha}Vß3 integrin was generated using two synthetic peptides containing the amino acid sequences encompassing positions 36–63 and 623–648. An antihuman ß3 subunit polyclonal antibody was purchased from Chemicon International (Temecula, CA). Antiphosphotyrosine (p-Tyr) and anti-IGF-IR antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). M1 anti-flag antibody was obtained from Sigma Chemical Co. (St. Louis, MO). The Src family kinase inhibitor PP2 was purchased from Calbiochem (San Diego, CA). EZ-link Sulfo NHS SS biotin was purchased from Pierce Chemical Co. (Rockford, IL). Vitronectin was purified from porcine serum using a modification of a published method (37).

pSMCs were prepared from porcine aortas as previously described (38). Cells were maintained in DMEM-H with 10% fetal bovine serum (Hyclone, Logan, UT) and streptomycin (100 µg/ml) and penicillin (100 U/ml). Cells that were between passages 4–16 were used in these experiments.

Generation of Expression Vectors and Establishment of the Cell Line
Full-length human ß3 integrin subunit that had been ligated into the pRcRSV vector was established previously (4). The pRcRSV-ß3 construct containing the two phenylalanine substitutions for Tyr 747 and 759 (ß3-FF) was generated using site-directed mutagenesis as described previously (4). To specifically detect the exogenous form of ß3, full-length ß3-FF was subcloned into expression vector pMEP4 containing a FLAG sequence (DYKDDDDK) after a ß3 leader sequence (pMEP4-ß3-FF/FLAG). The pMEP4-FLAG vector without the ß3-FF insert was used as a control. pSMCs (passages 4 and 5) were transfected with these constructs using the poly-L-ornithine method as described previously (38). Cells were trypsinized 48 h post transfection and seeded in three wells of a six-well dish and maintained in DMEM-H with 15% fetal bovine serum and hygromycin (40 µg/ml) plus streptomycin and penicillin. The hygromycin-resistant cells were grown to confluency and screened for expression of ß3-FF/FLAG. Three independent transfections for each construct were undertaken. The results presented are representative of between three and seven independent experiments. Each experiment was performed at least once using cells from two or three independent transfections. When the results obtained with cells from two independent transfections were compared, they were not significantly different. The cells used in these experiments were between passages 6 and 20. Expression of ß3-FF/FLAG protein was identified by immunoprecipitation with the M1 anti-FLAG antiserum followed by immunoblotting with the polyclonal anti-ß3 antiserum.

In some control experiments, cells were transfected with pMEP4-ß3 wild-type (WT) construct and selected in hygromycin containing media as described above. Since this construct does not encode FLAG sequence, a polyclonal antihuman ß3 antibody that does not cross-react with porcine ß3 was used to detect the exogenously expressed ß3-subunit. Cells transfected with ß3-WT or ß3-FF were grown to confluency, and cell surface proteins were biotinylated as previously described (4). Cells were then lysed and an equal amount of protein from each cell lysate was immunoprecipitated using a 1:300 dilution of the antihuman ß3 antibody. The immune complexes were then separated by 7.5% SDS-PAGE under nonreducing conditions. Biotinylated ß3 was detected by immunoblotting with peroxidase-conjugated ExtraAvidin as described below.

Our previous analysis of the integrin receptors expressed on the surface of pSMCs demonstrated that the ß3-subunit associates only with the {alpha}V- and not with other {alpha}-subunits (2). Because the {alpha}V-subunit also associates with ß5 in pSMCs, we used a ß3-specific antibody in all experiments to identify the {alpha}Vß3 integrin complex.

Immunoprecipitation and Immunoblotting
Cells were seeded at 5 x 106 cells per 10-cm plate (Beckton Dickinson Labware, Franklin Lakes, NJ) and grown for 7 d to reach confluency. Subconfluent cells were obtained after 3 d culture from the trypsinization. Before the initiation of the experiments, cultures were incubated in serum-free DMEM-H for 12–16 h after which IGF-I (100 ng/ml) was added directly to the medium for the times listed. In some experiments Src family kinase inhibitor PP2 (3.0 mM) was dissolved in dimethylsulfoxide (DMSO) and added to confluent cultures using increasing concentrations (2–8 µM) followed by incubation in serum-free medium overnight. Control cultures were exposed to DMSO alone, and it had no effect on ß3 phosphorylation. Vitronectin was added to a final concentration of 2 µg/ml and incubated with the cells for 2 h before lysis. The cell monolayers were lysed in ice-cold buffer (1% Triton X-100, 50 mM HEPES, 150 mM NaCl, 1 mM MgCl2, and 1 mM EGTA plus 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin, and the phosphatase inhibitors, sodium fluoride, 25 mM, and sodium orthovanadate, 2 mM). Total protein concentrations were determined using a BCA protein assay method (39) (Pierce Chemical Co.). The cell lysates were centrifuged at 14,000 x g for 10 min at 4 C, and the crude membrane extract was exposed to a 1:330 dilution of antiporcine ß3 antiserum overnight at 4 C. The immunoprecipitates were immobilized using protein-A sepharose beads for 2 h at 4 C and washed three times with the same buffer. The precipitated proteins were eluted in 40 µl of 2x Laemmeli sample buffer, boiled for 5 min, and separated on a 7.5% sodium dodecyl sulfate gel. The proteins were then transferred to Immobilon-P membrane and blocked for 1 h in 1% BSA in Tris-saline buffer with 0.1% Tween-20. The blots were incubated overnight at 4 C with indicated antibodies (1:500 for p-Tyr or anti-SHP-2 and 1:1000 for antibodies against IGF-IR and the ß3-subunit). Immunoprecipitation of the IGF-IR (1:300 dilution), SHPS-1 (1:300), and SHP-2 (1:300) was accomplished using the same general methods except that cell lysates were prepared using a modified 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 and phosphatase inhibitors described above. The proteins were detected using enhanced chemiluminescence (Pierce Chemical Co.) and analyzed using the GeneGnome CCD image system (Syngene, Ltd., Cambridge, UK). The images obtained were also scanned using an Agfa Scanner. Densitometric analyses of the images were determined using NIH Image, version 1.61.

Detection of SHP-2 in the Cytosol and Membrane Fractions
Confluent cells were washed twice with PBS and incubated with cell dissociation solution (Sigma, St. Louis, MO) for 10 min at room temperature. The cells were then scraped off the plate gently and transferred into centrifuge tubes. The samples were centrifuged at 1000 x g for 5 min to pellet the cells. The pellets were then resuspended in 900 µl of PBS with protease inhibitors (1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, and 1 µg/ml leupeptin) and stored at -80 C overnight. Cells were lysed by thawing at room temperature and sonicating for 2 min in a water bath. The samples were centrifuged at 14,000 x g for 10 min to pellet membrane-associated proteins. The supernatant containing cytosolic proteins was immunoprecipitated with anti-SHP-2 antibody using the conditions described above. The pellets were then resuspended in a buffer containing 0.2% Triton X-100, 150 mM NaCl, and 50 mM HEPES (pH 7.5), and the protease inhibitors listed above, and then centrifuged at 14,000 x g for 10 min. The supernatant containing Triton-soluble membrane-associated proteins was immunoprecipitated with anti-SHP-2 antibody. The precipitated SHP-2 was then separated from other proteins using 7.5% SDS-PAGE and detected by immunoblotting as described above.

Measurement of [3H]Thymidine Incorporation into pSMCs
pSMCs were plated at a density of 2.5 x 104/cm2 in 96-well tissue culture plates and grown for 5 d without changing the medium. The cultures were rinsed once with serum-free DMEM and incubated with DMEM plus 0.2% platelet-poor plasma for 24 h. The medium was changed to DMEM with 0.2% platelet-poor plasma, 0.1% DMSO containing various concentrations of IGF-I, and 0.5 µCi/well of [3H]thymidine (specific activity, 35 Ci/µmol). The cells were incubated at 37 C for 24 h and the amount of [3H]thymidine incorporated into DNA was determined as described previously (40).

Statistical Analysis
Student’s t test was used to compare the differences between control and treatment groups or control cells and cells expressing mutant ß3-FF. P < 0.05 was considered statistically significant.


    ACKNOWLEDGMENTS
 
We thank Ms. Laura Lindsey for assistance with the manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant AG-02331.

Abbreviations: ß3-FF, Mutant ß3 with two tyrosines changed to phenylalanine; ß3-WT, wild-type ß3; DMSO, dimethylsulfoxide; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate 1; PDGFR, platelet-derived growth factor receptor; PI-3 kinase, phosphatidylinositol 3 kinase; pSMCs, primary cultured smooth muscle cells; SMC, smooth muscle cell; SHPS-1, Src homology 2 domain-containing tyrosine phosphatase substrate 1.

Received for publication April 16, 2003. Accepted for publication May 27, 2003.


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