Recruitment of the Tyrosine Phosphatase Src Homology 2 Domain Tyrosine Phosphatase-2 to the p85 Subunit of Phosphatidylinositol-3
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《内分泌学杂志》
University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599
Abstract
IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.
Introduction
MULTIPLE GROWTH FACTORS and cytokines signal through the phosphatidylinositol-3 (PI-3) kinase pathway and several cellular processes that are stimulated by growth factors such as cellular replication, migration, increases in cell size, and inhibition of apoptosis are enhanced after PI-3 kinase activation (1, 2, 3, 4, 5, 6). The catalytic subunit of the class 1A PI-3 kinases (p110) catalyzes the conversion of membrane lipids to inositol triphosphate moieties in the plasma membrane, which results in membrane recruitment of multiple signaling molecules containing pleckstrin homology domains (7, 8). Several of these molecules have been shown to be important for signaling in the insulin/IGF-I pathway (9, 10, 11, 12, 13). The catalytic activity of the p110 subunit is activated by a conformational change that occurs when the regulatory subunit p85 binds to p110 (14). PI-3 kinase activity is further enhanced by p85 binding through its SH2 domains to phosphorylated tyrosines on growth factor receptors (15, 16). In the case of IGF-I and insulin receptors, PI-3 kinase activation occurs through binding of the p85/p110 complex to phosphorylated insulin receptor substrate (IRS)-1, a principal substrate of both these receptor tyrosine kinases (17, 18). Blocking PI-3 kinase activation has been shown in porcine smooth muscle cells (pSMCs) to markedly inhibit the ability of IGF-I to stimulate cell migration and to some extent cell proliferation (5).
Our laboratory and others have shown that IGF-I stimulation of smooth muscle cell (SMC) migration or proliferation requires ligand occupancy of the V3 integrin (19, 20). The mechanism by which ligand occupancy of the V3 integrin regulates IGF-I signaling and action is directly linked to the recruitment of the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 to downstream signaling molecules (19, 21). After ligand occupancy of V3, 3-subunit phosphorylation is stimulated, which results in SHP-2 recruitment to the plasma membrane. This membrane-associated SHP-2 is subsequently recruited to Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) whose tyrosines are phosphorylated in response to IGF-I receptor activation (19). Blocking SHP-2 recruitment to SHPS-1 results in impairment of IGF-I-stimulated cell migration (22) and expression of a phosphatase inactive form of SHP-2 mutant (SHP-2/C459S) has been associated with impaired breast cancer cell migration (21). Other investigators have shown that SHP-2 plays an important role in mediating cell spreading and migration in response to integrin, growth factor, or cytokine stimulation (23, 24, 25, 26).
Prior studies have shown that the association of SHP-2 with IRS-1 modulates the ability of epidermal growth factor (EGF), insulin, or IGF-I to activate PI-3 kinase (27, 28). A more direct role for SHP-2 in regulating PI-3 kinase was shown by Wu et al. (29), who showed that EGF induced p85 coimmunoprecipitation with SHP-2 in mouse fibroblasts and expression of a SHP-2 mutant with the N-terminal SH2 domain deletion resulted in impaired stimulation of PI-3 kinase and Akt phosphorylation in response to EGF, platelet-derived growth factor (PDGF), and IGF-I (19). Because the SH-2 domain in SHP-2 mediates its binding to target proteins (30, 31, 32), deletion of the SH2 domain could potentially disrupt SHP-2 recruitment to p85, thus leading to decrease PI kinase activity. The specific tyrosine motif that binds the SH2 domain of SHP-2 is the YxxI/L/V motif, which has been identified in SHPS-1 and IRS-1 (30, 31, 32). Growth factor stimulation induces phosphorylation of tyrosines contained in this motif in SHPS-1, leading to SHP-2 binding. In p85, tyrosines 528 and 556 are contained in these motifs, indicating that they have the potential to form binding sites for SHP-2. Although p85 undergoes tyrosine phosphorylation in response to PDGF and insulin, to date there is no published report regarding the function of these two specific tyrosines in growth factor signaling (33, 34). These studies were undertaken to determine whether SHP-2 binds to the p85 subunit of PI-3 kinase after IGF-I stimulation and whether disruption of SHP-2 transfer to p85 by mutating these two tyrosines would alter p85/p110 association, p85/p110 complex binding to IRS-1, and PI-3 kinase activation and thereby inhibit IGF-I-stimulated cell migration.
Materials and Methods
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 (Carlsbad, CA). Polyclonal antibodies for IRS-1, SHP-2, the p85 subunit, and the hemagglutinin epitope (HA) were obtained from Upstate Biotechnology (Lake Placid, NY). Antiphosphotyrosine (p-Tyr) and anti-p110 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-Akt or Akt were from BD Transduction Laboratories (Lexington, KY). Phosphatidylinositol substrate was from Avanti Polar Lipids (Alabaster, AL). Cold ATP was purchased from Sigma (St. Louis, MO) and [-32P]ATP was from Amersham Biosciences (Piscataway, NJ) . A synthetic peptide containing the TAT cell permeability sequence (35) followed by the Tyr556-containing motif (YXXI) of the p85 subunit YARAAARQARA555EYREIDKR562 (underlined and hereafter referred to as the p85 peptide) was synthesized by the Protein Chemistry Core Facility at the University of North Carolina at Chapel Hill. Purity and sequence confirmation were determined by mass spectrometry.
Cell culture
pSMCs were prepared from porcine aortas as previously described (36). Cells were maintained in DMEM-H with 10% fetal bovine serum (Hyclone, Logan, UT) and streptomycin (100 ng/ml) and penicillin (100 U/ml). The SMCs that were used in these experiments were used between passages 4 and 16.
Generation of HA-plenti-SHP-2 wild-type (WT) and HA-plenti-SHP-2/C459S
Full-length human SHP-2 cDNA was generated by RT-PCR from mRNA that had been derived from human fibroblasts (GM10, Human Genetic Cell Repository, Camden, NJ) (31) and cloned into the pEnter/D-TOPO Gateway entry vector according to the manufacturer’s instructions (Invitrogen). The forward and reverse primers used to generate the PCR product were: 5'-CACCATGACATCGCGGAGATGGTTTCACCC-3' and 5'-TTAAGCGTAATCTGGAACATCGTATGGGTATCTGAAACTT-TTCTGCTGTTGCATCAG-3'. The reverse primer contained the sequence encoding the HA epitope (underlined). The SHP-2 sequence was confirmed by DNA sequencing (UNC Genome Analysis Facility, Chapel Hill, NC) and was transferred from the entry-vector into pLenti6DV5 Gateway vector using the LR clonase reaction following manufacturer’s instructions (Invitrogen). The catalytically inactive SHP-2/C459S was generated by replacing cysteine 459 with serine in the catalytic domain of SHP-2 as described previously (31). The mutant was cloned into the entry-vector and subsequently transferred into the pLenti6DV5 vector as described for generation of SHP-2/WT.
Generation of HA-plenti6DV5, HA-plenti-p85WT (p85/WT), and HA-plenti p85/Y528F, Y556F.
The plenti6DV5 empty vector control was used as provided by the manufacturer (Invitrogen). Human p85DNA was obtained by PCR using first-strand cDNA transcribed from HepG2 cell total RNA. RNA (5 μg) was reverse transcribed using random hexamers and Superscript III (Invitrogen) at 50 C for 1 h. One microliter of the reaction mixture was used as template for PCR amplification using Advantage 2 polymerase (BD CLONTECH, Mountain View, CA). The forward and reverse primers were 5'-CACCatgagtgctgaggggtaccagtaca-3' and 5'-ttaAGCGTAATCTGGAACATCGTATGGGTAtcgcctctgctgtgcatatactgg-3'. A Kozac sequence was incorporated into the forward primer, and a sequence encoding the HA epitope was incorporated into the reverse primer. They are indicated by capital letters. The PCR product was cloned into pcDNA3.1V5His (Invitrogen). The p85 sequence was verified by DNA sequencing and subsequently cloned into the expression vector plenti-6DV5 using TOPO methodology (Invitrogen).
HA-plenti-p85/Y528F,Y556F mutant.
Substitutions for tyrosines 528 and 556 to phenylalanine were introduced into p85 by double-stranded mutagenesis. Three primers were used. The first encoded a base substitution to generate the Y528F (5'-gagacttcaacttatcaAaattatgcataatcctttg-3') and the second a base substitution to generate Y556F (5'-cgtttgtcaatttctcgaAactcagctgcctgctt-3'). The third oligonucleotide 5'-gctccttcggtcctcGAatTCttgtcagaagtaagt was used to change a unique PvuI site to a unique EcoRI site. All three oligonucleotide sequences were annealed to heat-denatured pcDNA3.1V5His-p85HA, extended using T4 DNA polymerase and ligated using T4 DNA ligase. The synthesis reaction product was digested with PvuI (to remove template) and the remaining wild-type/mutant hybrid was transformed into BMH71–18 bacteria (BD CLONETECH). The plasmid was isolated, digested with PvuI (to remove template), and transformed into TOP10 cells (Invitrogen). Clones were isolated and incorporation of the correct substitutions was verified by DNA sequencing. The p85 mutant was linearized with EcoRI and amplified by Pfx polymerase (Invitrogen). The blunt end product was directionally cloned into plenti6DV5 using TOPO methodology.
Establishment of SMCs expressing plenti6DV5 constructs
Transduction of pSMCs.
SMCs (passage 4–5) were seeded at 3 x 105/well in each of two wells of a 6-well plate (Falcon, no. 353046) the day before transduction. The viral stocks were thawed and the viral complexes precipitated as described previously (37). For transduction, the pellet was resuspended in 1 ml growth medium, 1 μl polybrene (40 mg/ml) was added, and the mixture was incubated with the cells for 24 h. The virus-containing medium was removed and changed to 2 ml growth medium for another 24 h and then replaced with selection medium (growth medium containing 4 μg/ml blasticidin). The cultures were then grown to confluency and expanded for experiments or frozen down for subsequent experiments. The expression of the HA-tagged p85/WT and p85 mutant proteins was detected by immunoblotting with a 1:1000 dilution of anti-HA antibody using 30 μl of cell lysate. The levels of exogenously expressed SHP-2 in each cell type were evaluated by immunoprecipitation with an anti-SHP-2 antibody followed by immunoblotting with an anti-HA antibody.
Immunoprecipitation and immunoblotting
Cells were seeded at 5 x 105 cells per 10-cm plate (Beckton Dickinson Labware, Franklin Lakes, NJ) and grown for 7 d to reach confluency. The cultures were incubated in serum-free DMEM-H for 12–16 h before the addition of IGF-I (100 ng/ml). In some experiments, nontransduced SMCs were preincubated with or without the synthetic peptide that contained a region of sequence from p85 for 1 h before IGF-I was added. 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 phenylmethylsulfonylfluoride, 1 μg/ml pepstatin), and phosphatase inhibitors (sodium fluoride, 25 mM, and sodium orthovanadate, 2 mM). The cell lysates were centrifuged at 14,000 x g for 10 min at 4 C. The supernatant containing crude membrane and cytosolic proteins was exposed to a 1:330 dilution of anti-IRS-1, anti-HA, anti-SHP-2, anti-p85, or anti-p110 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. The precipitated proteins were eluted in 40 μl of 2x Laemmeli sample buffer, boiled for 5 min, and separated on a 7.5% SDS-PAGE. The proteins were then transferred to Immobilon-P membranes that were blocked for 1 h in 1% BSA in Tris-saline buffer with 0.2%Tween 20. The blots were incubated overnight at 4 C with indicated antibody (1:500 for p-Tyr, 1:1000 for anti-HA, SHP-2, p85, p110, or IRS-1).
To detect phospho-Akt, 30 μl of cell lysate were removed before immunoprecipitation and mixed with 25 μl of 2x Lammaeli sample buffer and then separated by SDS-PAGE using an 8% gel. Antiphospho-Akt (1:1000) was used to detect phosphorylated Akt, and total Akt protein was detected using an anti-Akt antibody (1:1000). The proteins were detected using enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL) and their relative abundance analyzed using the GeneGnome CCD image system (Syngene Ltd., Cambridge, UK). The images obtained were also scanned using an DuoScan scanning densitometer (Agfa, Morstel, Belgium). Densitometric analyses of the images were determined using National Institutes of Health Image, version 1.61. To determine differences in AKT phosphorylation, arbitrary scanning units obtained for phospho-AKT band intensities were divided by arbitrary scanning units obtained for total AKT protein band intensities for each time point analyzed. The average values for each lane were obtained by pooling data from three separate experiments. All experiments were performed at least three times with similar results.
PI-3 kinase assay
The SMC cultures were serum starved overnight and stimulated with IGF-I (100 ng/ml) for 10 min before cells were lysed in the RIPA buffer. The cell lysates were prepared and the p85/p110 complex immunoprecipitated as described above in the immunoprecipitation and immunoblotting section. The PI-3 kinase immunoprecipitates were washed twice with lysis buffer containing 100 mM Tris-HCl (pH 7.5), 500 mM LiCl2, and 100 mM sodium vanadate and once with the reaction buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and 100 mM sodium vanadate). The pellet was resuspended in 50 μl of the reaction buffer, and the reaction was initiated by addition of 25 μl ATP mix containing 440 mM cold ATP, 30μCi [-32P]ATP, and 20 μg sonicated phosphatidylinositol (PI) substrate with 100 mM MgCl2. The samples were incubated for 10 min at 22 C, and 20 μl of 8 N HCl was added to stop the reaction. Then 160 μl chloroform/methanol (1:1) were added to extract the PI from the reaction mixture, after which 50 μl of the PI in the organic phase was spotted onto a TLC plate (Merck, Darmstadt, Germany) impregnated with 1% potassium oxylate and resolved by ascending chromatography (CHCl3:CH3OH:H2O:NH4OH at 60:47:11.3:2). The plates were dried and the lipid products were identified by autoradiography.
Cell wounding and migration assay
SMCs were plated in 6-well dishes and grown to confluency. A single-edge razor blade was used to scrape an area of cells, leaving a denuded area with a sharp visible demarcation wound line (11). The wounded monolayers were then rinsed twice in serum-free medium and incubated in serum-free medium containing 0.2% fetal bovine serum with or without IGF-I at 100 ng/ml for 48 h at 37 C. The cells were then fixed and stained (Diff quick; Dade Behring Inc., Newark, DE), and the number of cells that migrated into the wound area was counted. At least eight of the 10 previously selected 1-mm areas at the edge of the wound were counted for each data point. Each experiment was repeated three times, and the results are the means ± SE of eight determinations in each of the three separate experiments.
Statistical analysis
Student’s t test was used to compare the differences between the basal and IGF-I treatment, the control and peptide treatment groups, or control cells and cells expressing mutant proteins. P 0.05 was considered statistically significant.
Results
Expression of catalytic inactive SHP-2/C459S leads to decreases in IGF-I-dependent cell migration and phosphorylation of Akt
To determine whether SHP-2 had a role in mediating IGF-I-dependent cell migration, we compared the cell migratory responses to IGF-I in SMCs expressing either wild-type SHP-2 or the SHP-2/C459S mutant. IGF-I induced a 2.75 ± 0.21-fold increase in SMC migration in cells expressing WT-SHP-2 (mean ± SEM, n = 3, P < 0.05, compared with nonstimulated cultures), but the IGF-I-dependent cell migratory response was decreased to a 1.8 ± 0.07-fold increase (mean ± SEM, n = 3) in cells expressing SHP-2/C459S (Table 1) (P < 0.05, compared with the response of cells expressing wild-type SHP-2). Cells expressing vector alone showed a 2.3 ± 0.37-fold increase that was not different, compared with cells expressing WT-SHP-2. Because our prior studies (5) had shown that the activation of the PI-3 kinase pathway is primarily responsible for IGF-I-dependent cell migration, we determined the effect of expression of SHP-2/C459S on downstream signaling. Analysis of SHP-2 expression showed that similar levels of wild-type (HA-tagged) SHP-2 and SHP-2/C459S were expressed in SMCs (Fig. 1A). IGF-I induced phosphorylation of Akt in cells expressing WT-SHP-2. In contrast, after IGF-I stimulation, Akt phosphorylation normalized by Akt protein level was decreased by 47 ± 5, 55.6 ± 9, and 63.1 ± 6.2% (n = 3, mean ± SEM) at 5, 10, and 20 min, respectively, in cells expressing SHP-2/C459S (P < 0.05, compared with cells expressing WT-SHP-2) (Fig. 1B). Reprobing with an antibody that recognized total AKT showed that there were no significant differences and that changes in total AKT levels could not account for the differences in AKT phosphorylation (Fig. 1B). Therefore, expression of SHP-2/C459S resulted in decreased cell migration and impaired phosphorylation of Akt. These findings suggest that PI-3 kinase activation is also decreased.
Expression of SHP-2/C459S results in impaired association of SHP-2 with p85 and disruption of p85/p110 complex formation
We have previously shown that in cells expressing the SHP-2/C459S mutant, SHP-2 is recruited to its substrate protein SHPS-1 in response to IGF-I, but it does not dephosphorylate SHPS-1 and it is not transferred to other downstream signaling molecules (31). In some cell systems, the recruitment of SHP-2 to downstream signaling molecules is required for growth factor mediated cellular responses (19, 30, 38). Because activation of the PI-3 kinase pathway is impaired in SHP-2/C459S cells, we first examined whether SHP-2 and SHP-2/C459S could be recruited to the PI-3 kinase complex in response to IGF-I. As shown in Fig. 2, after IGF-I stimulation for 10 and 20 min, there were 2.7 ± 0.7- and 2.8 ± 0.8-fold increases in the association of HA-SHP-2/WT with p85 (n = 3, P < 0.05, compared with control cultures not exposed to IGF-I). However, in cells expressing SHP-2/C459S, no significant increase in IGF-I-induced SHP-2 association with p85 was detected (n = 3, 1.2 ± 0.2- and 0.8 ± 0.2-fold increases at 10 and 20 min, P = NS). Furthermore, in contrast to control cells that demonstrate constitutive binding of p85 and p110, the cells expressing SHP-2/C459S had disruption of p85/p110 complex formation, even though there was no change in the levels of p85 or p110 expression (Fig. 2). Hence, expression of SHP-2/C459S is associated with impaired recruitment of SHP-2 to p85 and dissociation of the p85/p110 complex.
Tyr528 and Tyr556 of the p85 subunit are required for SHP-2 binding in response to IGF-I
Because the phosphatase activity of SHP-2 and therefore its recruitment to multiple signaling proteins are impaired in the cells expressing SHP-2/C459S, it is not clear whether the impairment of the ability of IGF-I to activate signaling through PI-3 kinase in these cells is due to the loss of SHP-2 binding specifically to p85. To more precisely characterize the role of SHP-2 recruitment in mediating IGF-I-dependent PI-3 kinase activation, we generated a p85 mutant that had two tyrosines (528 and 556) substituted with phenylalanines. These two tyrosines are contained within YxxL/I/V motifs in p85 and therefore form potential SH2 domain recognition sites to which SHP-2 can bind. Analysis of the cell lysates by direct immunoblotting indicated that equivalent levels of HA-p85/WT and HA-p85/Y528F, Y556F were expressed after transduction (Fig. 3A). IGF-I stimulated a 3.4 ± 0.8-fold increase (n = 3, P < 0.05, compared with nonstimulated cultures) in SHP-2 binding to p85 in cells expressing p85WT, but this association was blocked in SMCs expressing the p85/Y528F, Y556F mutant (1.1 ± 0.5-fold increase, n = 3, P = NS, compared with nonstimulated cultures) (Fig. 3B). Furthermore, when nontransduced SMCs were preincubated with a cell permeable peptide that contains the sequence flanking tyrosine 556 in p85, the IGF-I-induced increase in SHP-2 binding to p85 was also inhibited (Fig. 3C). IGF-I induced a 2.8 ± 0.8-fold increase in SHP-2 binding to p85 in control cultures (n = 3, P < 0.05), but the response to IGF-I was decreased by 73.2 ± 5.6% in cultures exposed to the peptide (n = 3, P < 0.01, compared with the response of the control cultures).
Recruitment of SHP-2 to p85 is required for IGF-I-dependent activation of the PI-3 kinase and its downstream signaling
To determine whether loss of SHP-2 binding to p85 disrupted PI-3 kinase activation, we evaluated the PI-3 kinase activity in cells expressing p85Y528F, Y556F. After 10 min stimulation, IGF-I induced a 3.1 ± 0.8-fold increase in PI-3 kinase activity in the cells expressing p85WT (n = 3, P < 0.05), but there was no significant increase in cells expressing p85/Y528F, Y556F (1.2 ± 0.2-fold, n = 3, P = NS, compared with nonstimulated cultures) (Fig. 4A). To determine the consequences of the loss of PI-3 kinase activation, Akt phosphorylation was analyzed. IGF-I induced a 4.5 ± 1.2-fold increase (n = 3, P < 0.05, compared with nonstimulated cultures) in Akt phosphorylation in cells expressing WT/p85, and this response was decreased by 79.7 ± 13.3% in cells expressing p85/Y528F, Y556F (n = 3, mean ± SEM, P < 0.05, compared with cells expressing WT/p85) (Fig. 4B).
SHP-2 binding to p85 is required for IGF-I stimulated cell migration
Because PI-3 kinase activation is required for an optimal cell migration response to IGF-I, this response was compared in the two cell types. Cell migration was significantly impaired in SMCs expressing the p85 mutant. IGF-I induced a 2.06 ± 0.11-fold increase in migration in the cells expressing WT p85 (mean ± SEM n = 6, P < 0.01, compared with nonstimulated cultures). This is comparable with the 2.07 ± 0.14-fold increase in migration nontransfected cells. In contrast, this response was reduced to 1.12 ± 0.06-fold in the cells expressing this p85/Y528F, Y556F (mean ± SEM, n = 3, P < 0.05, compared with cells expressing WT p85) (Fig. 4C). When SMC migration was analyzed after exposure to the cell-permeable p85 peptide, the IGF-I-induced migratory response was decreased by 63.5 ± 5% (mean ± SEM, n = 6, P < 0.01, compared with control cultures not exposed to the peptide).
Effect of disruption of p85 binding to SHP-2 on IGF-I stimulated p85/p110 binding to IRS-1
To determine how loss of p85 binding to SHP-2 was leading to a decrease in PI-3 kinase activation, we first analyzed p85/p110 complex formation. In contrast to the cells expressing the SHP-2/C459S mutant, the p85/p110 complex was not disrupted in cells expressing the p85/Y528F, Y556F mutant (Fig. 5A). Cellular exposure to the p85 peptide that inhibited SHP-2 binding also did not disrupt p85/p110 complex formation (data not shown). Therefore, disruption of p85/SHP-2 binding alone does not interfere with constitutive association of p85 and p110. After IGF-I stimulation, the p85/p110 complex is recruited to tyrosine-phosphorylated IRS-1 (10, 39). This association leads to activation of the PI-3 kinase in response to IGF-I or insulin (9). To examine the effect of the loss of SHP-2 recruitment to p85 on the activation of the PI-3 kinase pathway, we examined the association of p85/p110 complex with IRS-1 in cells expressing wild-type p85 and in SMCs expressing the p85/Y528F, Y556F mutant. As shown in Fig. 5B, IGF-I induced a 3.7 ± 1.1-fold (mean ± SEM) increase in the association of p85/p110 with IRS-1 in cells expressing WT p85 (n = 3, P < 0.05, compared with nonstimulated cultures). However, this IGF-I-dependent increase in p85/p110 complex binding to IRS-1 was decreased by 50.5 ± 18.7% in cells expressing the p85/Y528F, Y556F mutant (n = 3, P < 0.05, compared with cells expressing WT p85). As a control, IGF-I-dependent IRS-1 phosphorylation was analyzed, and the results obtained using SMC that were expressing p85/Y528F, Y4556F showed no impairment (4.5 ± 0.4-fold increase in cells expressing p85/WT, compared with 5.2 ± 2.1-fold increase in cells expressing the p85 mutant, n = 3, P = NS).
Discussion
Previous studies have demonstrated both a positive and negative role for SHP-2 in regulating PI-3 kinase activation. Most of the studies have analyzed the EGF receptor linked signaling system. In general these studies have shown that EGF stimulates the association of SHP-2 with Gab-1 and p85-Gab-1 association (40, 41). These associations are often dependent on Gab-1 phosphorylation. Several of these studies have shown that formation of a complex between either Gab-1 or Gab-2 and SHP-2 or between Gab-1/Gab-2, and p85 is required for PI-3 kinase activation (41, 42). Takahashi et al. (43) demonstrated that both EGF and IGF-I stimulation of human skin fibroblasts resulted in the phosphorylation of two proteins (p115 and p105) and that these proteins associated with both SHP-2 and with p85. They proposed that these two proteins were platforms for recruiting p85, which simultaneously bound to SHP-2, and that such binding was required for full activation of PI-3 kinase activity.
In contrast to those studies, Zhang et al. (44) demonstrated that SHP-2 negatively regulated the strength and duration of PI-3 kinase activation in response to EGF receptor stimulation. Fibroblasts that expressed a phosphatase defective form of SHP-2 showed increased association of p85 with Gab-1 when compared with wild-type fibroblasts. They concluded that this was due to increased phosphorylation of the p85 binding sites on Gab-1. Therefore Gab-1-associated PI-3 kinase activity was increased, and PI-3 kinase downstream dependent signals were enhanced in the SHP-2 mutant cells after EGF stimulation. Based on these observations, they concluded that SHP-2 negatively regulated EGF-dependent PI-3 kinase activation by dephosphorylating the p85 binding sites on Gab1.
In addition to EGF, other growth factors including PDGF, insulin, and IGF-I have been shown to require SHP-2 for stimulation of PI-3 kinase activity. These studies have shown that disruption of SHP-2 phosphatase activity leads to impaired PI-3 kinase activation. Ugi et al. (28) demonstrated that expression of a mutant form of SHP-2 that lacked the phosphatase domain attenuated the ability of insulin to induce PI-3 kinase activation by modulating IRS-1 phosphorylation. They showed that in the presence of this mutant, there was reduced SHP-2 binding to IRS-1, which resulted in both attenuated insulin-stimulated IRS-1 phosphorylation and reduced activation of PI-3 kinase. Ivins Zito et al. (45) reported that overexpression of a catalytically inactive SHP-2 mutant inhibited IGF-I-dependent PI-3 kinase and Akt activation in fibroblasts and that expression of a SHP-2 mutant with a deletion of exon 3 resulted in the fibroblasts being hypersensitive to etoposide-induced cell death. This was rescued by reintroduction of wild-type SHP-2, suggesting a positive role of SHP-2 in IGF-I-mediated PI-3 kinase activation. However, none of these reports clearly delineated the molecular mechanism by which SHP-2 is functioning to enhance PI-3 kinase activity. Araki et al. (46) showed that phosphorylation of tyrosines 542 and 580 in SHP-2 is necessary for PDGF induction of MAPK; however, the substrates to which SHP-2 was binding to effect this change were not identified. Furthermore, studies that used SHP-2 mutants with the SH-2 domain deleted and studies using the phosphatase defective C459S mutant have not identified the specific target proteins to which SHP-2 needs to be recruited to activate PI-3 kinase.
In this study we made two important observations. First, we have shown that mutation of the catalytic domain of SHP-2 inhibits p85/p110 binding and subsequently PI-3 kinase activity. Second, we have shown that SHP-2 binding to p85 is required for p85/p110 complex association with IRS-1 in response to IGF-I, although it is not required for p85/p/110 association. Our results show that p85 binds to SHP-2 in response to IGF-I and that this interaction is modulated through two tyrosines that are contained in the YxxL/I/V motifs in p85. The tyrosines that are contained in this sequence motif in several substrates have been shown to mediate SHP-2 binding through its SH-2 domains (30, 31, 32). Although we did not directly demonstrate that IGF-I can induce phosphorylation of Y528, Y556, the observation that mutation of these tyrosines to phenylalanines disrupted SHP-2/p85 binding indirectly suggests that these tyrosines undergo phosphorylation and thus mediate SHP-2 binding to p85 in response to IGF-I. In our study, these mutations or an inhibitory peptide that disrupted the p85/SHP-2 interaction also resulted in failure to activate p85/p110 binding to IRS-1 or PI-3 kinase activity. A prior publication showed that the Grb10 associated with p85 via its SH2 domain in response to insulin stimulation (47); however, the binding site on p85 was not identified, Grb10/p85 binding did not alter p85 binding to IRS-1, and it was not involved in regulating IRS-1-associated PI-3 kinase activity. In this study we show that these tyrosines modulate SHP-2 binding to p85 and that the loss of SHP-2 binding is associated with the loss of p85/p110 association with IRS-1, resulting in failure to properly activate PI-3 kinase. This leads to attenuation of Akt activation. It remains to be determined why SHP-2 binding to p85 is required for its interaction with IRS-1. Because SHP-2 transfer to p85 and p85/p110 transfer to IRS-1 are required for PI kinase activation, this raises the question whether SHP-2 is mediating p85/p110 transfer. Myers et al. (32) showed that a mutation of IRS-1 that resulted in the loss of SHP-2 binding also resulted in enhanced p85 binding to IRS-1 in response to insulin. In addition, Luo et al. (48) showed that mutation of serine 1223 in IRS-1 resulted in increased SHP-2 association but decreased insulin-stimulated p85/IRS-1 association. These findings strongly suggest that SHP-2 is not directly mediating p85/p110 transfer to IRS-1.
Our studies do not resolve the issue of whether the full catalytic activity of SHP-2 is required for PI-3 kinase activation. Although we cannot exclude the possibility that coimmunoprecipitation is not sensitive enough to detect binding between SHP-2/C459S and p85, the difference in p85/SHP-2 binding between the cells expressing WT-SHP-2 and the SHP-2/C459S mutant suggests that even if the p85/SHP-2 interaction occurred, it has been significantly attenuated. Because mutation of the catalytic domain has been shown to result in the retention of SHP-2 on certain binding partners, it seems that this is the most likely explanation for the lack of SHP-2/p85 binding in the cells expressing SHP-2/ C459S.
Exactly how SHP-2 regulates p85/p110 binding and its activity has yet to be determined. Some studies have shown that dephosphorylation of p85 is required for optimal p110 activation; however, this has not been consistently demonstrated (49, 50). Although the SHP-2 phosphatase-deficient mutant has been used in several studies to analyze its function in mediating PI-3 kinase activation, a caveat in using this mutant is that after its recruitment to phosphorylated proteins, SHP-2 must dephosphorylate its binding partner to be recruited to downstream signaling intermediates (31). Because the phosphatase-deficient SHP-2 cannot dephosphorylate the binding partner, this makes it impossible to distinguish between the loss of phosphatase activity and the elimination of downstream protein/protein interactions. One other possibility is that another protein is binding to the p85/p110 complex that has to be dephosphorylated by SHP-2 for p85 to bind to p110.
Our previous studies have shown that blocking SHP-2 recruitment to the V3 integrin is associated with failure to transfer to SHP-2 to SHPS-1 and downstream signaling molecules (19). Blocking DOK1/3 association, which directly inhibits SHP-2 transfer to the 3-subunit of the V3 integrin, was associated with reduced activation of Akt in response to IGF-I (11). Because each of these manipulations should have led to failure to transfer SHP-2 to p85, the findings further support the conclusion that SHP-2 transfer is required for PI-3 kinase and Akt induction. Thus, stimulation of 3 integrin-linked signaling that allows SHP-2 recruitment to plasma membrane would optimally position cells to respond to IGF-I, which could then rapidly recruit SHP-2 to the p85 subunit either directly from the 3 subunit or indirectly via SHPS-1. Our findings also demonstrate that failure to recruit SHP-2 to p85 leads to inhibition of the association of the p85/p110 complex with IRS-1 but not to loss of IRS-1 phosphorylation. Because several studies have reported that recruitment of this complex to IRS-1 is necessary for IGF-I to stimulate PI-3 kinase activation, this may be the primary mechanism by which failure to transfer SHP-2 to p85 results in loss of PI-3 kinase activation. Other studies (48, 49, 51) have reported that p85 can activate other signaling mechanisms that are linked to IGF-I or insulin actions independently of PI-3 kinase activation. However, taken together with our previous report that activation of PI-3 kinase is required for IGF-I to stimulate cell migration, it seems most likely that blocking SHP-2 binding to p85 is inhibiting the cell migration response by disrupting the PI-3 kinase activation response to IGF-I. The mechanism by which SHP-2 recruitment to p85 facilitates p85/p110 recruitment to IRS-1 or whether it can directly activate PI-3 kinase should be the focus of further investigation.
Acknowledgments
The authors thank Ms. Laura Lindsey for her help in preparing the manuscript.
Footnotes
This work was supported by a grant from the National Institutes of Health (AG02331).
M.K., Y.L., L.A.M, and J.C. have nothing to declare. D.R.C. received consulting and lecture fees from Pfizer and consulting fees from Lilly.
First Published Online November 23, 2005
1 M.K. and Y.L. contributed equally to this work.
Abbreviations: DMEM-H, DMEM containing glucose/liter; EGF, epidermal growth factor; IRS, insulin receptor substrate; PDGF, platelet-derived growth factor; PI, phosphatidylinositol; PI-3 kinase, phosphatidylinositol 3 kinase; pSMC, porcine smooth muscle cell; p-Tyr, phosphotyrosine; RIPA, radioimmunoprecipitation assay; SHP-2, Src homology 2 domain tyrosine phosphatase; SHPS-1, Src homology 2 domain containing protein tyrosine phosphatase substrate-1; SMC, smooth muscle cell; WT, wild type.
Accepted for publication November 14, 2005.
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Abstract
IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.
Introduction
MULTIPLE GROWTH FACTORS and cytokines signal through the phosphatidylinositol-3 (PI-3) kinase pathway and several cellular processes that are stimulated by growth factors such as cellular replication, migration, increases in cell size, and inhibition of apoptosis are enhanced after PI-3 kinase activation (1, 2, 3, 4, 5, 6). The catalytic subunit of the class 1A PI-3 kinases (p110) catalyzes the conversion of membrane lipids to inositol triphosphate moieties in the plasma membrane, which results in membrane recruitment of multiple signaling molecules containing pleckstrin homology domains (7, 8). Several of these molecules have been shown to be important for signaling in the insulin/IGF-I pathway (9, 10, 11, 12, 13). The catalytic activity of the p110 subunit is activated by a conformational change that occurs when the regulatory subunit p85 binds to p110 (14). PI-3 kinase activity is further enhanced by p85 binding through its SH2 domains to phosphorylated tyrosines on growth factor receptors (15, 16). In the case of IGF-I and insulin receptors, PI-3 kinase activation occurs through binding of the p85/p110 complex to phosphorylated insulin receptor substrate (IRS)-1, a principal substrate of both these receptor tyrosine kinases (17, 18). Blocking PI-3 kinase activation has been shown in porcine smooth muscle cells (pSMCs) to markedly inhibit the ability of IGF-I to stimulate cell migration and to some extent cell proliferation (5).
Our laboratory and others have shown that IGF-I stimulation of smooth muscle cell (SMC) migration or proliferation requires ligand occupancy of the V3 integrin (19, 20). The mechanism by which ligand occupancy of the V3 integrin regulates IGF-I signaling and action is directly linked to the recruitment of the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 to downstream signaling molecules (19, 21). After ligand occupancy of V3, 3-subunit phosphorylation is stimulated, which results in SHP-2 recruitment to the plasma membrane. This membrane-associated SHP-2 is subsequently recruited to Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) whose tyrosines are phosphorylated in response to IGF-I receptor activation (19). Blocking SHP-2 recruitment to SHPS-1 results in impairment of IGF-I-stimulated cell migration (22) and expression of a phosphatase inactive form of SHP-2 mutant (SHP-2/C459S) has been associated with impaired breast cancer cell migration (21). Other investigators have shown that SHP-2 plays an important role in mediating cell spreading and migration in response to integrin, growth factor, or cytokine stimulation (23, 24, 25, 26).
Prior studies have shown that the association of SHP-2 with IRS-1 modulates the ability of epidermal growth factor (EGF), insulin, or IGF-I to activate PI-3 kinase (27, 28). A more direct role for SHP-2 in regulating PI-3 kinase was shown by Wu et al. (29), who showed that EGF induced p85 coimmunoprecipitation with SHP-2 in mouse fibroblasts and expression of a SHP-2 mutant with the N-terminal SH2 domain deletion resulted in impaired stimulation of PI-3 kinase and Akt phosphorylation in response to EGF, platelet-derived growth factor (PDGF), and IGF-I (19). Because the SH-2 domain in SHP-2 mediates its binding to target proteins (30, 31, 32), deletion of the SH2 domain could potentially disrupt SHP-2 recruitment to p85, thus leading to decrease PI kinase activity. The specific tyrosine motif that binds the SH2 domain of SHP-2 is the YxxI/L/V motif, which has been identified in SHPS-1 and IRS-1 (30, 31, 32). Growth factor stimulation induces phosphorylation of tyrosines contained in this motif in SHPS-1, leading to SHP-2 binding. In p85, tyrosines 528 and 556 are contained in these motifs, indicating that they have the potential to form binding sites for SHP-2. Although p85 undergoes tyrosine phosphorylation in response to PDGF and insulin, to date there is no published report regarding the function of these two specific tyrosines in growth factor signaling (33, 34). These studies were undertaken to determine whether SHP-2 binds to the p85 subunit of PI-3 kinase after IGF-I stimulation and whether disruption of SHP-2 transfer to p85 by mutating these two tyrosines would alter p85/p110 association, p85/p110 complex binding to IRS-1, and PI-3 kinase activation and thereby inhibit IGF-I-stimulated cell migration.
Materials and Methods
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 (Carlsbad, CA). Polyclonal antibodies for IRS-1, SHP-2, the p85 subunit, and the hemagglutinin epitope (HA) were obtained from Upstate Biotechnology (Lake Placid, NY). Antiphosphotyrosine (p-Tyr) and anti-p110 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-Akt or Akt were from BD Transduction Laboratories (Lexington, KY). Phosphatidylinositol substrate was from Avanti Polar Lipids (Alabaster, AL). Cold ATP was purchased from Sigma (St. Louis, MO) and [-32P]ATP was from Amersham Biosciences (Piscataway, NJ) . A synthetic peptide containing the TAT cell permeability sequence (35) followed by the Tyr556-containing motif (YXXI) of the p85 subunit YARAAARQARA555EYREIDKR562 (underlined and hereafter referred to as the p85 peptide) was synthesized by the Protein Chemistry Core Facility at the University of North Carolina at Chapel Hill. Purity and sequence confirmation were determined by mass spectrometry.
Cell culture
pSMCs were prepared from porcine aortas as previously described (36). Cells were maintained in DMEM-H with 10% fetal bovine serum (Hyclone, Logan, UT) and streptomycin (100 ng/ml) and penicillin (100 U/ml). The SMCs that were used in these experiments were used between passages 4 and 16.
Generation of HA-plenti-SHP-2 wild-type (WT) and HA-plenti-SHP-2/C459S
Full-length human SHP-2 cDNA was generated by RT-PCR from mRNA that had been derived from human fibroblasts (GM10, Human Genetic Cell Repository, Camden, NJ) (31) and cloned into the pEnter/D-TOPO Gateway entry vector according to the manufacturer’s instructions (Invitrogen). The forward and reverse primers used to generate the PCR product were: 5'-CACCATGACATCGCGGAGATGGTTTCACCC-3' and 5'-TTAAGCGTAATCTGGAACATCGTATGGGTATCTGAAACTT-TTCTGCTGTTGCATCAG-3'. The reverse primer contained the sequence encoding the HA epitope (underlined). The SHP-2 sequence was confirmed by DNA sequencing (UNC Genome Analysis Facility, Chapel Hill, NC) and was transferred from the entry-vector into pLenti6DV5 Gateway vector using the LR clonase reaction following manufacturer’s instructions (Invitrogen). The catalytically inactive SHP-2/C459S was generated by replacing cysteine 459 with serine in the catalytic domain of SHP-2 as described previously (31). The mutant was cloned into the entry-vector and subsequently transferred into the pLenti6DV5 vector as described for generation of SHP-2/WT.
Generation of HA-plenti6DV5, HA-plenti-p85WT (p85/WT), and HA-plenti p85/Y528F, Y556F.
The plenti6DV5 empty vector control was used as provided by the manufacturer (Invitrogen). Human p85DNA was obtained by PCR using first-strand cDNA transcribed from HepG2 cell total RNA. RNA (5 μg) was reverse transcribed using random hexamers and Superscript III (Invitrogen) at 50 C for 1 h. One microliter of the reaction mixture was used as template for PCR amplification using Advantage 2 polymerase (BD CLONTECH, Mountain View, CA). The forward and reverse primers were 5'-CACCatgagtgctgaggggtaccagtaca-3' and 5'-ttaAGCGTAATCTGGAACATCGTATGGGTAtcgcctctgctgtgcatatactgg-3'. A Kozac sequence was incorporated into the forward primer, and a sequence encoding the HA epitope was incorporated into the reverse primer. They are indicated by capital letters. The PCR product was cloned into pcDNA3.1V5His (Invitrogen). The p85 sequence was verified by DNA sequencing and subsequently cloned into the expression vector plenti-6DV5 using TOPO methodology (Invitrogen).
HA-plenti-p85/Y528F,Y556F mutant.
Substitutions for tyrosines 528 and 556 to phenylalanine were introduced into p85 by double-stranded mutagenesis. Three primers were used. The first encoded a base substitution to generate the Y528F (5'-gagacttcaacttatcaAaattatgcataatcctttg-3') and the second a base substitution to generate Y556F (5'-cgtttgtcaatttctcgaAactcagctgcctgctt-3'). The third oligonucleotide 5'-gctccttcggtcctcGAatTCttgtcagaagtaagt was used to change a unique PvuI site to a unique EcoRI site. All three oligonucleotide sequences were annealed to heat-denatured pcDNA3.1V5His-p85HA, extended using T4 DNA polymerase and ligated using T4 DNA ligase. The synthesis reaction product was digested with PvuI (to remove template) and the remaining wild-type/mutant hybrid was transformed into BMH71–18 bacteria (BD CLONETECH). The plasmid was isolated, digested with PvuI (to remove template), and transformed into TOP10 cells (Invitrogen). Clones were isolated and incorporation of the correct substitutions was verified by DNA sequencing. The p85 mutant was linearized with EcoRI and amplified by Pfx polymerase (Invitrogen). The blunt end product was directionally cloned into plenti6DV5 using TOPO methodology.
Establishment of SMCs expressing plenti6DV5 constructs
Transduction of pSMCs.
SMCs (passage 4–5) were seeded at 3 x 105/well in each of two wells of a 6-well plate (Falcon, no. 353046) the day before transduction. The viral stocks were thawed and the viral complexes precipitated as described previously (37). For transduction, the pellet was resuspended in 1 ml growth medium, 1 μl polybrene (40 mg/ml) was added, and the mixture was incubated with the cells for 24 h. The virus-containing medium was removed and changed to 2 ml growth medium for another 24 h and then replaced with selection medium (growth medium containing 4 μg/ml blasticidin). The cultures were then grown to confluency and expanded for experiments or frozen down for subsequent experiments. The expression of the HA-tagged p85/WT and p85 mutant proteins was detected by immunoblotting with a 1:1000 dilution of anti-HA antibody using 30 μl of cell lysate. The levels of exogenously expressed SHP-2 in each cell type were evaluated by immunoprecipitation with an anti-SHP-2 antibody followed by immunoblotting with an anti-HA antibody.
Immunoprecipitation and immunoblotting
Cells were seeded at 5 x 105 cells per 10-cm plate (Beckton Dickinson Labware, Franklin Lakes, NJ) and grown for 7 d to reach confluency. The cultures were incubated in serum-free DMEM-H for 12–16 h before the addition of IGF-I (100 ng/ml). In some experiments, nontransduced SMCs were preincubated with or without the synthetic peptide that contained a region of sequence from p85 for 1 h before IGF-I was added. 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 phenylmethylsulfonylfluoride, 1 μg/ml pepstatin), and phosphatase inhibitors (sodium fluoride, 25 mM, and sodium orthovanadate, 2 mM). The cell lysates were centrifuged at 14,000 x g for 10 min at 4 C. The supernatant containing crude membrane and cytosolic proteins was exposed to a 1:330 dilution of anti-IRS-1, anti-HA, anti-SHP-2, anti-p85, or anti-p110 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. The precipitated proteins were eluted in 40 μl of 2x Laemmeli sample buffer, boiled for 5 min, and separated on a 7.5% SDS-PAGE. The proteins were then transferred to Immobilon-P membranes that were blocked for 1 h in 1% BSA in Tris-saline buffer with 0.2%Tween 20. The blots were incubated overnight at 4 C with indicated antibody (1:500 for p-Tyr, 1:1000 for anti-HA, SHP-2, p85, p110, or IRS-1).
To detect phospho-Akt, 30 μl of cell lysate were removed before immunoprecipitation and mixed with 25 μl of 2x Lammaeli sample buffer and then separated by SDS-PAGE using an 8% gel. Antiphospho-Akt (1:1000) was used to detect phosphorylated Akt, and total Akt protein was detected using an anti-Akt antibody (1:1000). The proteins were detected using enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL) and their relative abundance analyzed using the GeneGnome CCD image system (Syngene Ltd., Cambridge, UK). The images obtained were also scanned using an DuoScan scanning densitometer (Agfa, Morstel, Belgium). Densitometric analyses of the images were determined using National Institutes of Health Image, version 1.61. To determine differences in AKT phosphorylation, arbitrary scanning units obtained for phospho-AKT band intensities were divided by arbitrary scanning units obtained for total AKT protein band intensities for each time point analyzed. The average values for each lane were obtained by pooling data from three separate experiments. All experiments were performed at least three times with similar results.
PI-3 kinase assay
The SMC cultures were serum starved overnight and stimulated with IGF-I (100 ng/ml) for 10 min before cells were lysed in the RIPA buffer. The cell lysates were prepared and the p85/p110 complex immunoprecipitated as described above in the immunoprecipitation and immunoblotting section. The PI-3 kinase immunoprecipitates were washed twice with lysis buffer containing 100 mM Tris-HCl (pH 7.5), 500 mM LiCl2, and 100 mM sodium vanadate and once with the reaction buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and 100 mM sodium vanadate). The pellet was resuspended in 50 μl of the reaction buffer, and the reaction was initiated by addition of 25 μl ATP mix containing 440 mM cold ATP, 30μCi [-32P]ATP, and 20 μg sonicated phosphatidylinositol (PI) substrate with 100 mM MgCl2. The samples were incubated for 10 min at 22 C, and 20 μl of 8 N HCl was added to stop the reaction. Then 160 μl chloroform/methanol (1:1) were added to extract the PI from the reaction mixture, after which 50 μl of the PI in the organic phase was spotted onto a TLC plate (Merck, Darmstadt, Germany) impregnated with 1% potassium oxylate and resolved by ascending chromatography (CHCl3:CH3OH:H2O:NH4OH at 60:47:11.3:2). The plates were dried and the lipid products were identified by autoradiography.
Cell wounding and migration assay
SMCs were plated in 6-well dishes and grown to confluency. A single-edge razor blade was used to scrape an area of cells, leaving a denuded area with a sharp visible demarcation wound line (11). The wounded monolayers were then rinsed twice in serum-free medium and incubated in serum-free medium containing 0.2% fetal bovine serum with or without IGF-I at 100 ng/ml for 48 h at 37 C. The cells were then fixed and stained (Diff quick; Dade Behring Inc., Newark, DE), and the number of cells that migrated into the wound area was counted. At least eight of the 10 previously selected 1-mm areas at the edge of the wound were counted for each data point. Each experiment was repeated three times, and the results are the means ± SE of eight determinations in each of the three separate experiments.
Statistical analysis
Student’s t test was used to compare the differences between the basal and IGF-I treatment, the control and peptide treatment groups, or control cells and cells expressing mutant proteins. P 0.05 was considered statistically significant.
Results
Expression of catalytic inactive SHP-2/C459S leads to decreases in IGF-I-dependent cell migration and phosphorylation of Akt
To determine whether SHP-2 had a role in mediating IGF-I-dependent cell migration, we compared the cell migratory responses to IGF-I in SMCs expressing either wild-type SHP-2 or the SHP-2/C459S mutant. IGF-I induced a 2.75 ± 0.21-fold increase in SMC migration in cells expressing WT-SHP-2 (mean ± SEM, n = 3, P < 0.05, compared with nonstimulated cultures), but the IGF-I-dependent cell migratory response was decreased to a 1.8 ± 0.07-fold increase (mean ± SEM, n = 3) in cells expressing SHP-2/C459S (Table 1) (P < 0.05, compared with the response of cells expressing wild-type SHP-2). Cells expressing vector alone showed a 2.3 ± 0.37-fold increase that was not different, compared with cells expressing WT-SHP-2. Because our prior studies (5) had shown that the activation of the PI-3 kinase pathway is primarily responsible for IGF-I-dependent cell migration, we determined the effect of expression of SHP-2/C459S on downstream signaling. Analysis of SHP-2 expression showed that similar levels of wild-type (HA-tagged) SHP-2 and SHP-2/C459S were expressed in SMCs (Fig. 1A). IGF-I induced phosphorylation of Akt in cells expressing WT-SHP-2. In contrast, after IGF-I stimulation, Akt phosphorylation normalized by Akt protein level was decreased by 47 ± 5, 55.6 ± 9, and 63.1 ± 6.2% (n = 3, mean ± SEM) at 5, 10, and 20 min, respectively, in cells expressing SHP-2/C459S (P < 0.05, compared with cells expressing WT-SHP-2) (Fig. 1B). Reprobing with an antibody that recognized total AKT showed that there were no significant differences and that changes in total AKT levels could not account for the differences in AKT phosphorylation (Fig. 1B). Therefore, expression of SHP-2/C459S resulted in decreased cell migration and impaired phosphorylation of Akt. These findings suggest that PI-3 kinase activation is also decreased.
Expression of SHP-2/C459S results in impaired association of SHP-2 with p85 and disruption of p85/p110 complex formation
We have previously shown that in cells expressing the SHP-2/C459S mutant, SHP-2 is recruited to its substrate protein SHPS-1 in response to IGF-I, but it does not dephosphorylate SHPS-1 and it is not transferred to other downstream signaling molecules (31). In some cell systems, the recruitment of SHP-2 to downstream signaling molecules is required for growth factor mediated cellular responses (19, 30, 38). Because activation of the PI-3 kinase pathway is impaired in SHP-2/C459S cells, we first examined whether SHP-2 and SHP-2/C459S could be recruited to the PI-3 kinase complex in response to IGF-I. As shown in Fig. 2, after IGF-I stimulation for 10 and 20 min, there were 2.7 ± 0.7- and 2.8 ± 0.8-fold increases in the association of HA-SHP-2/WT with p85 (n = 3, P < 0.05, compared with control cultures not exposed to IGF-I). However, in cells expressing SHP-2/C459S, no significant increase in IGF-I-induced SHP-2 association with p85 was detected (n = 3, 1.2 ± 0.2- and 0.8 ± 0.2-fold increases at 10 and 20 min, P = NS). Furthermore, in contrast to control cells that demonstrate constitutive binding of p85 and p110, the cells expressing SHP-2/C459S had disruption of p85/p110 complex formation, even though there was no change in the levels of p85 or p110 expression (Fig. 2). Hence, expression of SHP-2/C459S is associated with impaired recruitment of SHP-2 to p85 and dissociation of the p85/p110 complex.
Tyr528 and Tyr556 of the p85 subunit are required for SHP-2 binding in response to IGF-I
Because the phosphatase activity of SHP-2 and therefore its recruitment to multiple signaling proteins are impaired in the cells expressing SHP-2/C459S, it is not clear whether the impairment of the ability of IGF-I to activate signaling through PI-3 kinase in these cells is due to the loss of SHP-2 binding specifically to p85. To more precisely characterize the role of SHP-2 recruitment in mediating IGF-I-dependent PI-3 kinase activation, we generated a p85 mutant that had two tyrosines (528 and 556) substituted with phenylalanines. These two tyrosines are contained within YxxL/I/V motifs in p85 and therefore form potential SH2 domain recognition sites to which SHP-2 can bind. Analysis of the cell lysates by direct immunoblotting indicated that equivalent levels of HA-p85/WT and HA-p85/Y528F, Y556F were expressed after transduction (Fig. 3A). IGF-I stimulated a 3.4 ± 0.8-fold increase (n = 3, P < 0.05, compared with nonstimulated cultures) in SHP-2 binding to p85 in cells expressing p85WT, but this association was blocked in SMCs expressing the p85/Y528F, Y556F mutant (1.1 ± 0.5-fold increase, n = 3, P = NS, compared with nonstimulated cultures) (Fig. 3B). Furthermore, when nontransduced SMCs were preincubated with a cell permeable peptide that contains the sequence flanking tyrosine 556 in p85, the IGF-I-induced increase in SHP-2 binding to p85 was also inhibited (Fig. 3C). IGF-I induced a 2.8 ± 0.8-fold increase in SHP-2 binding to p85 in control cultures (n = 3, P < 0.05), but the response to IGF-I was decreased by 73.2 ± 5.6% in cultures exposed to the peptide (n = 3, P < 0.01, compared with the response of the control cultures).
Recruitment of SHP-2 to p85 is required for IGF-I-dependent activation of the PI-3 kinase and its downstream signaling
To determine whether loss of SHP-2 binding to p85 disrupted PI-3 kinase activation, we evaluated the PI-3 kinase activity in cells expressing p85Y528F, Y556F. After 10 min stimulation, IGF-I induced a 3.1 ± 0.8-fold increase in PI-3 kinase activity in the cells expressing p85WT (n = 3, P < 0.05), but there was no significant increase in cells expressing p85/Y528F, Y556F (1.2 ± 0.2-fold, n = 3, P = NS, compared with nonstimulated cultures) (Fig. 4A). To determine the consequences of the loss of PI-3 kinase activation, Akt phosphorylation was analyzed. IGF-I induced a 4.5 ± 1.2-fold increase (n = 3, P < 0.05, compared with nonstimulated cultures) in Akt phosphorylation in cells expressing WT/p85, and this response was decreased by 79.7 ± 13.3% in cells expressing p85/Y528F, Y556F (n = 3, mean ± SEM, P < 0.05, compared with cells expressing WT/p85) (Fig. 4B).
SHP-2 binding to p85 is required for IGF-I stimulated cell migration
Because PI-3 kinase activation is required for an optimal cell migration response to IGF-I, this response was compared in the two cell types. Cell migration was significantly impaired in SMCs expressing the p85 mutant. IGF-I induced a 2.06 ± 0.11-fold increase in migration in the cells expressing WT p85 (mean ± SEM n = 6, P < 0.01, compared with nonstimulated cultures). This is comparable with the 2.07 ± 0.14-fold increase in migration nontransfected cells. In contrast, this response was reduced to 1.12 ± 0.06-fold in the cells expressing this p85/Y528F, Y556F (mean ± SEM, n = 3, P < 0.05, compared with cells expressing WT p85) (Fig. 4C). When SMC migration was analyzed after exposure to the cell-permeable p85 peptide, the IGF-I-induced migratory response was decreased by 63.5 ± 5% (mean ± SEM, n = 6, P < 0.01, compared with control cultures not exposed to the peptide).
Effect of disruption of p85 binding to SHP-2 on IGF-I stimulated p85/p110 binding to IRS-1
To determine how loss of p85 binding to SHP-2 was leading to a decrease in PI-3 kinase activation, we first analyzed p85/p110 complex formation. In contrast to the cells expressing the SHP-2/C459S mutant, the p85/p110 complex was not disrupted in cells expressing the p85/Y528F, Y556F mutant (Fig. 5A). Cellular exposure to the p85 peptide that inhibited SHP-2 binding also did not disrupt p85/p110 complex formation (data not shown). Therefore, disruption of p85/SHP-2 binding alone does not interfere with constitutive association of p85 and p110. After IGF-I stimulation, the p85/p110 complex is recruited to tyrosine-phosphorylated IRS-1 (10, 39). This association leads to activation of the PI-3 kinase in response to IGF-I or insulin (9). To examine the effect of the loss of SHP-2 recruitment to p85 on the activation of the PI-3 kinase pathway, we examined the association of p85/p110 complex with IRS-1 in cells expressing wild-type p85 and in SMCs expressing the p85/Y528F, Y556F mutant. As shown in Fig. 5B, IGF-I induced a 3.7 ± 1.1-fold (mean ± SEM) increase in the association of p85/p110 with IRS-1 in cells expressing WT p85 (n = 3, P < 0.05, compared with nonstimulated cultures). However, this IGF-I-dependent increase in p85/p110 complex binding to IRS-1 was decreased by 50.5 ± 18.7% in cells expressing the p85/Y528F, Y556F mutant (n = 3, P < 0.05, compared with cells expressing WT p85). As a control, IGF-I-dependent IRS-1 phosphorylation was analyzed, and the results obtained using SMC that were expressing p85/Y528F, Y4556F showed no impairment (4.5 ± 0.4-fold increase in cells expressing p85/WT, compared with 5.2 ± 2.1-fold increase in cells expressing the p85 mutant, n = 3, P = NS).
Discussion
Previous studies have demonstrated both a positive and negative role for SHP-2 in regulating PI-3 kinase activation. Most of the studies have analyzed the EGF receptor linked signaling system. In general these studies have shown that EGF stimulates the association of SHP-2 with Gab-1 and p85-Gab-1 association (40, 41). These associations are often dependent on Gab-1 phosphorylation. Several of these studies have shown that formation of a complex between either Gab-1 or Gab-2 and SHP-2 or between Gab-1/Gab-2, and p85 is required for PI-3 kinase activation (41, 42). Takahashi et al. (43) demonstrated that both EGF and IGF-I stimulation of human skin fibroblasts resulted in the phosphorylation of two proteins (p115 and p105) and that these proteins associated with both SHP-2 and with p85. They proposed that these two proteins were platforms for recruiting p85, which simultaneously bound to SHP-2, and that such binding was required for full activation of PI-3 kinase activity.
In contrast to those studies, Zhang et al. (44) demonstrated that SHP-2 negatively regulated the strength and duration of PI-3 kinase activation in response to EGF receptor stimulation. Fibroblasts that expressed a phosphatase defective form of SHP-2 showed increased association of p85 with Gab-1 when compared with wild-type fibroblasts. They concluded that this was due to increased phosphorylation of the p85 binding sites on Gab-1. Therefore Gab-1-associated PI-3 kinase activity was increased, and PI-3 kinase downstream dependent signals were enhanced in the SHP-2 mutant cells after EGF stimulation. Based on these observations, they concluded that SHP-2 negatively regulated EGF-dependent PI-3 kinase activation by dephosphorylating the p85 binding sites on Gab1.
In addition to EGF, other growth factors including PDGF, insulin, and IGF-I have been shown to require SHP-2 for stimulation of PI-3 kinase activity. These studies have shown that disruption of SHP-2 phosphatase activity leads to impaired PI-3 kinase activation. Ugi et al. (28) demonstrated that expression of a mutant form of SHP-2 that lacked the phosphatase domain attenuated the ability of insulin to induce PI-3 kinase activation by modulating IRS-1 phosphorylation. They showed that in the presence of this mutant, there was reduced SHP-2 binding to IRS-1, which resulted in both attenuated insulin-stimulated IRS-1 phosphorylation and reduced activation of PI-3 kinase. Ivins Zito et al. (45) reported that overexpression of a catalytically inactive SHP-2 mutant inhibited IGF-I-dependent PI-3 kinase and Akt activation in fibroblasts and that expression of a SHP-2 mutant with a deletion of exon 3 resulted in the fibroblasts being hypersensitive to etoposide-induced cell death. This was rescued by reintroduction of wild-type SHP-2, suggesting a positive role of SHP-2 in IGF-I-mediated PI-3 kinase activation. However, none of these reports clearly delineated the molecular mechanism by which SHP-2 is functioning to enhance PI-3 kinase activity. Araki et al. (46) showed that phosphorylation of tyrosines 542 and 580 in SHP-2 is necessary for PDGF induction of MAPK; however, the substrates to which SHP-2 was binding to effect this change were not identified. Furthermore, studies that used SHP-2 mutants with the SH-2 domain deleted and studies using the phosphatase defective C459S mutant have not identified the specific target proteins to which SHP-2 needs to be recruited to activate PI-3 kinase.
In this study we made two important observations. First, we have shown that mutation of the catalytic domain of SHP-2 inhibits p85/p110 binding and subsequently PI-3 kinase activity. Second, we have shown that SHP-2 binding to p85 is required for p85/p110 complex association with IRS-1 in response to IGF-I, although it is not required for p85/p/110 association. Our results show that p85 binds to SHP-2 in response to IGF-I and that this interaction is modulated through two tyrosines that are contained in the YxxL/I/V motifs in p85. The tyrosines that are contained in this sequence motif in several substrates have been shown to mediate SHP-2 binding through its SH-2 domains (30, 31, 32). Although we did not directly demonstrate that IGF-I can induce phosphorylation of Y528, Y556, the observation that mutation of these tyrosines to phenylalanines disrupted SHP-2/p85 binding indirectly suggests that these tyrosines undergo phosphorylation and thus mediate SHP-2 binding to p85 in response to IGF-I. In our study, these mutations or an inhibitory peptide that disrupted the p85/SHP-2 interaction also resulted in failure to activate p85/p110 binding to IRS-1 or PI-3 kinase activity. A prior publication showed that the Grb10 associated with p85 via its SH2 domain in response to insulin stimulation (47); however, the binding site on p85 was not identified, Grb10/p85 binding did not alter p85 binding to IRS-1, and it was not involved in regulating IRS-1-associated PI-3 kinase activity. In this study we show that these tyrosines modulate SHP-2 binding to p85 and that the loss of SHP-2 binding is associated with the loss of p85/p110 association with IRS-1, resulting in failure to properly activate PI-3 kinase. This leads to attenuation of Akt activation. It remains to be determined why SHP-2 binding to p85 is required for its interaction with IRS-1. Because SHP-2 transfer to p85 and p85/p110 transfer to IRS-1 are required for PI kinase activation, this raises the question whether SHP-2 is mediating p85/p110 transfer. Myers et al. (32) showed that a mutation of IRS-1 that resulted in the loss of SHP-2 binding also resulted in enhanced p85 binding to IRS-1 in response to insulin. In addition, Luo et al. (48) showed that mutation of serine 1223 in IRS-1 resulted in increased SHP-2 association but decreased insulin-stimulated p85/IRS-1 association. These findings strongly suggest that SHP-2 is not directly mediating p85/p110 transfer to IRS-1.
Our studies do not resolve the issue of whether the full catalytic activity of SHP-2 is required for PI-3 kinase activation. Although we cannot exclude the possibility that coimmunoprecipitation is not sensitive enough to detect binding between SHP-2/C459S and p85, the difference in p85/SHP-2 binding between the cells expressing WT-SHP-2 and the SHP-2/C459S mutant suggests that even if the p85/SHP-2 interaction occurred, it has been significantly attenuated. Because mutation of the catalytic domain has been shown to result in the retention of SHP-2 on certain binding partners, it seems that this is the most likely explanation for the lack of SHP-2/p85 binding in the cells expressing SHP-2/ C459S.
Exactly how SHP-2 regulates p85/p110 binding and its activity has yet to be determined. Some studies have shown that dephosphorylation of p85 is required for optimal p110 activation; however, this has not been consistently demonstrated (49, 50). Although the SHP-2 phosphatase-deficient mutant has been used in several studies to analyze its function in mediating PI-3 kinase activation, a caveat in using this mutant is that after its recruitment to phosphorylated proteins, SHP-2 must dephosphorylate its binding partner to be recruited to downstream signaling intermediates (31). Because the phosphatase-deficient SHP-2 cannot dephosphorylate the binding partner, this makes it impossible to distinguish between the loss of phosphatase activity and the elimination of downstream protein/protein interactions. One other possibility is that another protein is binding to the p85/p110 complex that has to be dephosphorylated by SHP-2 for p85 to bind to p110.
Our previous studies have shown that blocking SHP-2 recruitment to the V3 integrin is associated with failure to transfer to SHP-2 to SHPS-1 and downstream signaling molecules (19). Blocking DOK1/3 association, which directly inhibits SHP-2 transfer to the 3-subunit of the V3 integrin, was associated with reduced activation of Akt in response to IGF-I (11). Because each of these manipulations should have led to failure to transfer SHP-2 to p85, the findings further support the conclusion that SHP-2 transfer is required for PI-3 kinase and Akt induction. Thus, stimulation of 3 integrin-linked signaling that allows SHP-2 recruitment to plasma membrane would optimally position cells to respond to IGF-I, which could then rapidly recruit SHP-2 to the p85 subunit either directly from the 3 subunit or indirectly via SHPS-1. Our findings also demonstrate that failure to recruit SHP-2 to p85 leads to inhibition of the association of the p85/p110 complex with IRS-1 but not to loss of IRS-1 phosphorylation. Because several studies have reported that recruitment of this complex to IRS-1 is necessary for IGF-I to stimulate PI-3 kinase activation, this may be the primary mechanism by which failure to transfer SHP-2 to p85 results in loss of PI-3 kinase activation. Other studies (48, 49, 51) have reported that p85 can activate other signaling mechanisms that are linked to IGF-I or insulin actions independently of PI-3 kinase activation. However, taken together with our previous report that activation of PI-3 kinase is required for IGF-I to stimulate cell migration, it seems most likely that blocking SHP-2 binding to p85 is inhibiting the cell migration response by disrupting the PI-3 kinase activation response to IGF-I. The mechanism by which SHP-2 recruitment to p85 facilitates p85/p110 recruitment to IRS-1 or whether it can directly activate PI-3 kinase should be the focus of further investigation.
Acknowledgments
The authors thank Ms. Laura Lindsey for her help in preparing the manuscript.
Footnotes
This work was supported by a grant from the National Institutes of Health (AG02331).
M.K., Y.L., L.A.M, and J.C. have nothing to declare. D.R.C. received consulting and lecture fees from Pfizer and consulting fees from Lilly.
First Published Online November 23, 2005
1 M.K. and Y.L. contributed equally to this work.
Abbreviations: DMEM-H, DMEM containing glucose/liter; EGF, epidermal growth factor; IRS, insulin receptor substrate; PDGF, platelet-derived growth factor; PI, phosphatidylinositol; PI-3 kinase, phosphatidylinositol 3 kinase; pSMC, porcine smooth muscle cell; p-Tyr, phosphotyrosine; RIPA, radioimmunoprecipitation assay; SHP-2, Src homology 2 domain tyrosine phosphatase; SHPS-1, Src homology 2 domain containing protein tyrosine phosphatase substrate-1; SMC, smooth muscle cell; WT, wild type.
Accepted for publication November 14, 2005.
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