Receptor protein-tyrosine phosphatase α regulates focal adhesion kinase phosphorylation and ErbB2 oncoprotein-mediated mammary epithelial cell motility.

Background: PTP ! has been implicated in breast cancer, but its function remains to be defined. Results: Suppression of PTP ! led to a GRB7-dependent, ErbB2-mediated increase in mammary epithelial cell migration. PTP ! dephosphorylated FAK on Tyr-407. Conclusion: PTP ! functions to suppress ErbB2 signaling events that lead to migration of breast cancer cells. Significance: PTP ! may play positive or negative roles in signaling, depending upon context. We investigated the role of protein-tyrosine phosphatase ! (PTP ! ) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTP ! , suggesting that attenuation of PTP ! activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTP ! ledtoincreasedcellmigrationinanErbB2-dependentman-ner. The ability of ErbB2 to increase cell motility in the absence of PTP ! was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a " activation.

most of the catalytic activity. Uniquely to PTP␣, the membrane-distal D2 domain is also active, but with lower specific activity than D1. Furthermore, D2 appears to play a role in sensing reactive oxygen species (14,15) and, following oxidation, may participate in "inside-out" signaling by altering the rotational coupling of PTP␣ molecules within a receptor dimer (16). There is considerable evidence supporting a role for PTP␣ in activating SRC and other SRC family kinases (13,(17)(18)(19). However, the biological activity of PTPs is highly context-dependent (20), and it is possible that PTP␣ may recognize other physiological substrates. In fact, p130 cas (21), Kv1.2 (22), and the insulin receptor (23) have also been proposed to be substrates of PTP␣.
The ability of PTP␣ to activate SRC family kinases is the mechanism by which this receptor-like phosphatase transforms rat embryo fibroblasts (17). On this basis, it has been assumed that PTP␣ functions positively to promote tumorigenesis. Consistent with this, PTP␣ is overexpressed in latestage colon carcinoma (24), oral squamous carcinoma (25), and gastric carcinoma (26). Nevertheless, the situation is actually more complex. Expression of PTP␣ varies widely in breast cancer. In some cases, high levels of PTP␣ are associated with low tumor grade and reduced aggressiveness (27). In addition, metastasizing breast tumors (stage 3) were reported to express low levels of PTP␣ (27). Consistent with this, ectopic expression of PTP␣ in ErbB2-positive human mammary tumor cells reduces tumor growth and delays lung metastasis (27). In contrast, experiments in MMTV-ErbB2/PTP␣ Ϫ/Ϫ mice suggest that ablation of PTP␣ does not contribute to ErbB2-induced mammary tumor initiation or metastasis (28). In light of these apparently conflicting observations, this study was designed to address the function of PTP␣ in ErbB2-induced signaling in human mammary epithelial cells.
Generation of FLAG-tagged PTP␣ Fusion Proteins-Fulllength Human PTP␣ was cloned into p3XFLAG-CMV-13 mammalian expression vector (Sigma, catalog no. E4776), in which the N-terminal preprotrypsin leader sequence preceding the multiple cloning region was deleted. Using p3XFLAG-CMV13-PTP␣ (WT) as the template, expression constructs for trapping mutants (PTP␣ (D1 DA ) and PTP␣ (D2 EA )) were generated by QuikChange mutagenesis. All these constructs have a C-terminal 3XFLAG tag.
Hydrogen Peroxide Molecular Imaging-Molecular imaging of ErbB2-induced hydrogen peroxide production in 10A.B2 cells was studied using a PerkinElmer Life Sciences Ultraview spinning disk confocal operating on a Nikon Ti microscope with the in vivo scientific chamber, heater, and gas regulator as described previously (30). Images were analyzed using ImageJ (Wayne Rasband, National Institutes of Health).
Assay of PTP Oxidation-In PTPs, the catalytic cysteinyl residue is present as a thiolate anion in resting cells. After ErbB2 activation by AP1510, the cells were lysed in a degassed buffer at pH 5.5 containing iodoacetic acid. The active-site cysteinyl residue of PTPs that remained in a reduced state was terminally inactivated by alkylation. Conversely, the active-site cysteines of PTPs that were oxidized by second-messenger reactive oxygen species molecules were protected from irreversible alkylation. Iodoacetic acid was then removed from the lysate by size exclusion chromatography, and the reversibly oxidized activesite cysteinyl residues were reduced back to the thiolate ion with Tris(2-carboxyethyl)phosphine (TCEP). PTPs were maintained in pH 5.5 buffers and incubated with a biotinylated sulfhydryl-reactive iodoacetyl-PEG2 probe. After purification by streptavidin pull-down, PTPs that were oxidized in response to ErbB2 signaling were identified by immunoblotting (31).
Generation of Cells Expressing shRNA PTP␣-For stable PTP␣ knockdown in 10A.B2 cells, we expressed a pMLP retroviral vector in a pMSCV backbone (32) using the targeting sequence CAGATGGTGCAAACCGATA incorporated into the sequence of the human microRNA-30 (miR30). The infected cells were selected in medium containing 1-2 g of puromycin, and EGFP coexpression was verified using a Zeiss Axiovert 200 M microscope.
Immunoprecipitation and Immunoblotting-HA-ErbB2, tyrosine-phosphorylated proteins, and FAK were immunoprecipitated as follows. Cells were grown to 90% confluence in 10-cm plates, serum-starved for 16 h, and stimulated with AP1510 to induce ErbB2 dimerization and activation for the indicated times. After treatment, the cells were washed with cold PBS and extracted in 800 l of a lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 20 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 20 mM NaF, 1 mM PMSF, and protease inhibitor mixture. All subsequent steps were carried out on ice or at 4°C. Cells were lysed on a rotating wheel at 4°C for 30 min. Cell debris were centrifuged at 12,000 ϫ g for 10 min, and protein concentrations were determined. An equal amount of protein was diluted in cold lysis buffer and precleared for 60 min with protein A/G-Sepharose. The supernatants were first incubated for 60 min on a rotating wheel with appropriate antibodies, and 10 l of protein A/G-Sepharose was then added for another 60 min. The immune complexes were pelleted at 3000 ϫ g for 5 min and washed three times with lysis buffer. The beads were resuspended in 20 l of 4ϫ Laemmli sample buffer and heated at 95°C for 1 min. Proteins were separated by SDS-PAGE and detected by immunoblotting.
Cell Migration Assays-Cell motility was studied using a Boyden chamber-based migration assay (33) using cell culture inserts (8.0-m pore size) for 6-well plates (BD Falcon). For siRNA studies, knockdowns were performed with specified siRNAs (si␣-1, 5Ј-CAGAUGGUGCAAACCGAUA dTdT-3Ј; si␣-2, 5Ј-AAGCUGGGAGCCAUUCCAAUU dTdT-3Ј) using Lipofectamine as described (34). To quantitate cell motility, 100,000 cells were seeded on the inserts. After 48 h, the cells were washed with 1ϫ PBS and fixed with 5% buffered Formalin solution, stained, and counted. The cells retained inside the insert were removed, and those that had migrated through the pores to the bottom surface of the transwell were counted. For each condition, the number of migrating cells was counted in eight random microscopic fields. The number of migrating cells in the control 10A.B2 cells without stimulation was normalized to 1. Where indicated, AP1510 (1 M), G7-18NATE-Penetratin (G7-18NATE-P) peptide (GRB7 inhibitory peptide, WFEGYDNTFPC-RQIKIWFQNRRMKWKK) or Penetratin (RQIKIWFQNRRMKWKK) were added to the culture medium at the beginning of the assay. Cell motility was quantitated after 48 h.
In Vitro Phosphatase Assay-HA-tagged PTP␣ was expressed in HEK293T cells, purified, and washed several times with icecold reducing buffer (50 mM HEPES (pH7.4), 100 mM NaCl, 0.1% Triton X-100, 2 mM DTT, and a protease inhibitors tablet) for 10 min on ice to complete the reduction of PTP␣. The reduced enzyme was then incubated with phosphorylated FAK at 30°C for 30 min. The reaction was stopped by addition of 20 l of 4ϫ Laemmli sample buffer and heated at 95°C for 1 min. Proteins were separated by SDS-PAGE, and substrate dephosphorylation was visualized by immunoblotting.

RESULTS
Cooperation between PTP␣ and ErbB2 Signaling in Mammary Epithelial Cells-We tested the effects of suppressing the expression of PTP␣ on ErbB2-induced signaling in mammary epithelial cells using two independent siRNA sequences. The effect of PTP␣ suppression on ErbB2-induced cell motility was examined using a Boyden chamber-based migration assay. Dimerization and activation of ErbB2 was induced in MCF10A cells that expressed a well characterized chimeric form of the kinase (10A.B2) the activity of which can be induced by addition of a small molecule dimerizer, AP1510, as described previously (35). Activation of ErbB2 in parental 10A.B2 cells or cells treated with scrambled siRNA resulted in an ϳ3.5-fold stimulation of migration. Following treatment with an siRNA sequence shown previously to suppress PTP␣ (34) and a second siRNA designed using the RNAi Codex program at Cold Spring Harbor Laboratory, we observed that ErbB2 activation resulted in an 6-to 8-fold increase in cell motility compared with the basal migration observed in unstimulated 10A.B2 cells (Fig.  1A). Consistent with this observation, both siRNAs efficiently suppressed PTP␣ expression, whereas the nonspecific siRNA did not (Fig. 1B).
A stable cell line expressing the most effective shRNA targeting PTP␣ was then established, and cells were selected. Following the selection, the depletion of PTP␣ by shRNA in 10A.B2 cells was estimated to be ϳ90%. Specificity in the effect of the shRNA was illustrated by the fact that expression of the closely related PTP family member PTP⑀ was not affected (Fig. 1C). Using these shRNA-expressing 10A.B2 cells, the effect of PTP␣ suppression on ErbB2-induced cell motility was then re-examined. In the presence of AP1510, the migration of parental 10A.B2 cells was increased 3-fold. In contrast, following shRNA-depletion of PTP␣, ErbB2 activation resulted in a 5-fold increase in cell motility compared with the basal migration of unstimulated 10A.B2 cells (Fig. 1D). Hence, attenuation of PTP␣ contributed to increased ErbB2 signaling.
Transient Oxidation and Inactivation of PTP␣ in Response to ErbB2 Signaling in Mammary Epithelial Cells-Hydrogen peroxide (H 2 O 2 ) has been shown to inactivate protein-tyrosine phosphatases and, thereby, to promote protein-tyrosine phosphorylation-dependent signal transduction (36). Suppressing the expression of a particular PTP effectively reproduces oxidation-mediated inactivation and increases the phosphorylation of sites that are targeted by that PTP to promote downstream events in the signaling cascade (37). To determine whether PTP␣ was reversibly oxidized in the context of ErbB2 signaling, first we measured H 2 O 2 production using molecular imaging with a specific fluorescence indicator, PF6-AM (30). This probe features an aryl boronate chemical switch that allows for selective detection of H 2 O 2 over other reactive oxygen species molecules (38 -41). Following serum withdrawal, 10A.B2 cells were loaded with PF6-AM and treated with AP1510 ( Fig. 2A). ErbB2 activation caused a rapid and time-dependent increase in intracellular fluorescence. Considering the known selectivity of PF6- AM, our data indicate that, following addition of AP1510 and ErbB2 dimerization in 10A.B2 mammary epithelial cells, endogenous generation of H 2 O 2 occurred in less than 2 min and peaked at 5 min. In addition, preincubation of 10A.B2 cells with a chemical inhibitor of NADPH oxidases, diphenyleneiodonium (DPI), prevented ErbB2-mediated H 2 O 2 production and intracellular fluorescence of PF6-AM.
We tested whether DPI compromised signaling downstream of ErbB2, as reported previously for other receptor tyrosine kinases (31,42). Serum-starved cells were stimulated with AP1510 in the presence or absence of DPI. Cellular extracts were prepared, and tyrosine phosphorylated proteins were visualized by anti-pTyr immunoprecipitation and immunoblotting. Interestingly, inhibition of H 2 O 2 production by DPI led to decreased tyrosine phosphorylation of proteins of ϳ180, 75, 55, and 45 kDa (Fig. 2B). In addition, blotting with anti-HA revealed that the ϳ180-kDa band comigrated with ErbB2. Hence, decreasing the acute generation of H 2 O 2 , which would be expected to increase the level of active PTPs, compromised tyrosine phosphorylation and activation of ErbB2 and its downstream targets in mammary epithelial cells.
We have shown previously that PTP␣ is reversibly oxidized upon EGF receptor activation in IMR90 fibroblasts (43). Hence, we used a modified cysteinyl-labeling assay (43) to ascertain whether PTP␣ activity was also regulated by H 2 O 2 generated upon ErbB2 activation in 10A.B2 cells. This is a three-step method in which the reversibly oxidized invariant catalytic cysteine residue is specifically biotinylated by a thiolate anion-directed probe (Fig. 2C). Serum-starved 10A.B2 cells were incubated with AP1510 and lysed in an anaerobic work station that was purged and constantly supplied with ultrapure argon gas to prevent post-lysis oxidation of PTPs. We detected only minimal biotin labeling of PTP␣ in untreated cells. However, the biotinylation pattern from the cysteinyl labeling assay revealed that the reversible oxidation of PTP␣ occurred in a biphasic manner upon ErbB2 activation (Fig. 2D). Thus, ErbB2 activation attenuated PTP␣ activity by reversible oxidation in mammary epithelial cells.
Identification of Focal Adhesion Kinase as a Substrate of PTP␣-To identify substrates of PTP␣ that were components of ErbB2-induced signaling pathways, we treated 10A.B2 cells with AP1510 to activate ErbB2 and pervanadate to amplify the signal of potential phosphotyrosine-containing substrates for analysis. Substrate-trapping mutant forms of PTP␣, PTP␣D1 DA , and PTP␣D2 EA (Fig. 3A) were then utilized to identify potential physiological substrates of the PTP, as described previously (34). Proteins involved in ErbB2 signaling and cell migration were tested as potential interacting partners of PTP␣(WT), PTP␣(D1 DA ), or PTP␣(D2 EA ). Interestingly, the PTK FAK was enriched with PTP␣-trapping mutants (Fig. 3B). Considering previous studies implicating FAK in ErbB2-induced cell migration, invasion, and focal adhesion turnover (44,45), we investigated this novel PTP␣-FAK interaction further.
To determine whether the interaction of FAK with the PTP␣-trapping mutant occurred via the PTP active site, the trapping experiment was performed in the presence of sodium orthovanadate, a competitive inhibitor and transition state analog of phosphate that prevents substrate binding (46). The interaction of FAK with PTP␣(D1 DA ) was inhibited by vanadate, indicating the involvement of the active site and suggest-  DECEMBER 27, 2013 • VOLUME 288 • NUMBER 52

JOURNAL OF BIOLOGICAL CHEMISTRY 36929
ing that FAK may represent a direct substrate of PTP␣ in cells (Fig. 3C). Under the conditions of our substrate-trapping experiments, it is possible that multiprotein complexes containing the mutant PTP may be isolated. Therefore, we investigated whether PTP␣ could dephosphorylate FAK directly in vitro. FAK was immunoprecipitated from lysates of 10A.B2 cells treated with AP1510 and pervanadate. Under these conditions, FAK was phosphorylated on Tyr-397, Tyr-407, Tyr-576, Tyr-861, and Tyr-925 (Fig. 3D). The immunoprecipitates of phosphorylated FAK were then incubated with wild-type active PTP␣, and dephosphorylation was measured using phosphospecific anti-pTyr antibodies. Immunoblot analyses revealed that PTP␣ could dephosphorylate phospho-Tyr-407 of FAK specifically in vitro, whereas none of the other Tyr residues was dephosphorylated following incubation with the phosphatase. A role of PTP␣ in regulating the phosphorylation status of FAK Tyr-407 was verified in parental 10A.B2 cells and in 10A.B2 cells stably expressing shRNA for PTP␣. By immunoblotting using a phosphosite-specific antibody, we observed transient phosphorylation of Tyr-407 of FAK 30 -45 min following ErbB2 activation with AP1510 (Fig. 3E). In contrast, FAK Tyr-407 phosphorylation was sustained from 15-60 min following ErbB2 activation when PTP␣ expression was compromised. Collectively, these results suggest a novel role for PTP␣ in regulating phosphorylation of FAK on Tyr-407.
Interaction of FAK pTyr-407 with Vinculin-Tyrosine phosphorylation of FAK by SRC and PYK2 (proline-rich tyrosine kinase 2) leads to its activation and association with several SH2 domain-containing proteins as well as with focal adhesion proteins such as paxillin and vinculin. Previous studies in human ventricular endothelial cells have shown that phosphorylation of FAK on tyrosine 407 by PYK2 led to the recruitment of FAK to vinculin and paxillin (47,48). Hence, to understand the role of PTP␣ in dephosphorylating FAK, we tested whether FAK pTyr-407 behaved similarly in mammary epithelial cells. FAK was immunoprecipitated from 10A.B2 cells that were incubated with AP1510 for 30 min. We observed FAK interaction with paxillin and vinculin in 10A.B2 cells when PTP␣ was suppressed. However, vinculin was not coimmunoprecipitated with FAK in parental 10A.B2 cells (Fig. 4). In addition, because  FAK interacts directly with GRB7 (growth factor receptor bound 7) to promote cell migration (49), we also tested for the presence of GRB7 and its interacting partner ErbB2 (50) in FAK complexes. Both GRB7 and ErbB2 were detected in FAK complexes upon ErbB2 activation, independently of PTP␣ expression. This shows that the hyperphosphorylation of FAK Tyr-407 observed in the absence of PTP␣ contributed to the recruitment of vinculin to FAK in a multiprotein complex.
Increased Association of ␤1-Integrin and GRB7 with ErbB2 upon Suppression of PTP␣-A significant body of evidence indicates that the presence of vinculin in focal adhesions is critical for integrin-mediated cell adhesion and migration (reviewed in Ref. 51). ␤1-integrin is required for proliferation, survival, and invasiveness of human breast cancer cell lines (52). Integrins associate with the EGF receptor (53,54), and ErbB2 transactivation is impaired in ␤1-integrin-deficient breast tumors (55). Considering that an ErbB2-vinculin complex was detected in the absence of PTP␣ and that vinculin is recruited to the cytoplasmic tails of ␤-integrins (56), we tested whether the increased ErbB2-mediated migration observed in the absence of PTP␣ coincided with a change in the formation of an ErbB2-␤1-integrin complex. We monitored the association of ␤1-integrin and GRB7 with ErbB2. ErbB2 was immunoprecipitated following activation in intact 10A.B2 cells, or in cells in which PTP␣ expression was compromised, and immunoblotted for interacting proteins (Fig. 5). The interaction of PTP␣, GRB7, and ␤1-integrin with ErbB2 was transient and peaked between 30 -45 min in parental 10A.B2 cells, whereas suppression of PTP␣ levels resulted in a rapid and sustained association of ␤1-integrin with ErbB2. Hence, ErbB2-dependent migration in the absence of PTP␣ is likely to coincide with enhanced signaling of the receptor at ␤1-integrin-rich focal adhesions.
Decreased PTP␣ Expression Led to Enhanced ErbB2-GRB7 Interaction and GRB7-dependent Cell Migration-The GRB7 gene, encoding the SH2-containing adaptor protein GRB7, is part of the ERBB2 amplicon, an ϳ86,000-bp region that includes six genes (TCAP, PNMT, PERLD1, HER2, C17orf37/ C35, and GRB7) that is amplified in breast cancer (57). It has been reported that GRB7 is present at focal adhesions (58) regulating motility and tumorigenesis in cancer cells (59). GRB7 was present in FAK and ErbB2 immunoprecipitates following the activation of ErbB2 by AP1510. Hence, the presence of GRB7 in the ErbB2-FAK-␤1-integrin complexes prompted us to investigate the relationship between PTP␣, GRB7, and ErbB2 as part of a potential mechanism leading to increased motility of 10A.B2 cells. To this effect, ErbB2 was activated, immunoprecipitated from 10A.B2 cells expressing shRNA for PTP␣ or from parental 10A.B2 cells, and then the immunoprecipitates were probed for the presence of the GRB7 adaptor. Consistent with previous observations (50), GRB7 associated with ErbB2 (Fig. 6A). In 10A.B2 cells, there was a low basal level of association, with gradual GRB7 recruitment to ErbB2 upon receptor FIGURE 5. Effect of PTP␣ suppression on ErbB2 interaction with a ␤1-integrin complex. Serum-deprived 10A.B2 and 10A.B2-shPTP␣ cells plated on fibronectin (25 g/ml) were treated with 1 M AP1510 for the indicated times. HA-ErbB2 was immunoprecipitated (IP) from 100 g of cell lysate using anti-HA and immunoblotted using anti-␤1-integrin, anti-HA, or anti-GRB7 antibodies. ErbB2 was detected using anti-HA antibody. activation. In contrast, following PTP␣ knockdown, the basal level of interaction was increased, and ErbB2-induced association of GRB7 to ErbB2 occurred more rapidly (Fig. 6A).
To examine the importance of the ErbB2-GRB7 interaction on cell motility observed in the absence of PTP␣, we tested the effects of a GRB7 inhibitor on ErbB2-induced migration. We used a non-phosphorylated inhibitor peptide specific for the GRB7 SH2 domain, G7-18NATE (GRB7-peptide18-No Arms Thioether, Fig. 6B) bound to a penetratin peptide (G7-18NA-TE-P) (29) that has been shown previously to attenuate the migration of pancreatic cancer cells (60). The effect of inhibiting GRB7 on ErbB2-induced cell motility in 10A.B2 cells stably expressing shRNA for PTP␣ was examined using a Boyden chamber-based migration assay. ErbB2 activation resulted in a 5-fold increase in cell migration compared with the basal migration observed in unstimulated 10A.B2 cells. However, treatment of these PTP␣-depleted cells with AP1510 in the presence of the GRB7 inhibitor G7-18NATE-P abolished ErbB2-induced cell motility, whereas incubation with the penetratin peptide alone had no effect (Fig. 6C). This suggests that the increased interaction between GRB7 and ErbB2 observed in the absence of PTP␣ led to a GRB7-dependent increase in 10A.B2 cell migration.

DISCUSSION
Although PTP␣ has the capacity to display oncogenic properties, its biological role in mammary epithelial cells and breast cancer is unclear (27,34,61 and reviewed in Ref. 4). It has been shown that PTP␣ expression levels vary widely among breast tumors. Furthermore, it is unclear whether PTP␣ plays a positive or negative role in signaling in breast cancer (27). In this study, we examined the role of PTP␣ in ErbB2 signaling using a chimeric form of the kinase that could be induced by addition of a small-molecule dimerizing agent, AP1510, in human mammary epithelial 10A.B2 cells. We found that PTP␣ is a negative regulator of ErbB2-dependent 10A.B2 cell motility. In addressing the function of PTP␣ in ErbB2 signaling, we uncovered a novel function of the phosphatase in regulating the phosphorylation of FAK on tyrosine 407, regulating FAK binding to vinculin and prolonging the association of ErbB2 with GRB7 and ␤1-integrins. In addition, PTP␣-mediated, ErbB2-dependent cell motility was also dependent upon GRB7 acting as an ErbB2-interacting protein. The consequences of RNAi-induced suppression of PTP␣ suggest an important role for this receptor protein-tyrosine phosphatase in controlling ErbB2 signal transduction, leading to migration of human mammary cancer cells.
We have shown previously that PTP␣ is reversibly oxidized following EGF receptor activation (43). Others have also observed a role of PTP␣ in EGF receptor signaling in diverse mechanisms, such as aging (62), as well as in cell-substratum adhesion (63). However, this is the first study implicating PTP␣ in the regulation of ErbB2-mediated cell motility. We utilized siRNA targeting human PTP␣, designed using the RNAi Codex program at Cold Spring Harbor Laboratory, and confirmed the migration phenotype with another siRNA sequence shown previously to be a potent suppressor of PTP␣ expression in the Shalloway laboratory (34). By repressing PTP␣ expression, which would mimic the oxidation-mediated reversible inactivation of the enzyme that occurs in signaling in cells, we increased the phosphorylation of sites targeted by PTP␣. This suggested that the transient inactivation of PTP␣ may control the phosphorylation of FAK and the formation of GRB7 complexes involved in the migratory phenotype. Reversible oxidation of the catalytic cysteine of the D1 domain would be expected to inactivate its function directly. Furthermore, reversible oxidation of the D2 domain of PTP␣ has been shown to cause the formation of a disulfide bond with the catalytic cysteine of the counterpart D2 domain in the dimer, thereby inducing a conformational change and inhibition of the D1 domain (reviewed in Ref. 6). Hence, the reversible oxidation of either the D1 or D2 cysteine of PTP␣, as detected by the cysteinyl labeling assay, is a measure of the inactivation of PTP␣ occurring following the rapid increase in intracellular hydrogen peroxide that takes place upon acute ErbB2 activation.
The highly dynamic process of cell migration, regulated by tyrosine phosphorylation within focal complexes, involves modulation of cell-substrate adhesion and recruitment of over 50 structural proteins to the cytoplasmic segments of ␣-and ␤-integrins (64). FAK is a central regulator of focal complexes. It has been implicated in cancer cell motility in vitro in addition to being an important contributor to tumor invasion, metastasis, and malignancy (65)(66)(67). There have been reports indicating that, under certain circumstances, PTP␣, acting via stimulation of SRC, may promote phosphorylation of Tyr-397 in FAK (68). In this study, analysis by RNAi-induced suppression of PTP␣, application of substrate-trapping mutant forms of the enzyme, and a direct phosphatase activity assay all illustrate dephosphorylation of Tyr-407 of FAK by PTP␣. We did not observe significant changes in any other sites of tyrosine phosphorylation in FAK, highlighting the specificity of the phosphatase. Moreover, we found that FAK Tyr-407 phosphorylation was prolonged upon ErbB2 activation when PTP␣ expression was compromised. FAK Tyr-407 has been shown previously to be phosphorylated by PYK2 in response to VEGF stimulation (48) as well as by SRC (69). In addition to its function as a kinase, phosphorylation of FAK is known to promote its function as a scaffold protein (49). We have observed that FAKpTyr-407 displayed preferential recruitment to vinculin in addition to being associated with ErbB2, GRB7, and paxillin. This pTyr-407-dependent interaction between FAK and vinculin has also been observed by others (47). However, the significance of pTyr-407 phosphorylation is still unclear. Previous groups have shown that FAK Tyr-407 phosphorylation occurs at focal adhesions and at the periphery of migrating cells (48,70), in tumor cell differentiation (71), and in epithelial mesenchymal transdifferentiation (70). Interestingly, it has also been proposed to be a FAK regulatory site (72). Thus, because ErbB2-induced cell migration, invasion, and focal adhesion turnover is dependent on FAK signaling (44,45), identifying the SH2-containing signaling protein bound to phosphoTyr-407 in this context may yield further insight into the role of PTP␣ in the ErbB2-dependent migration of human mammary epithelial cells.
Our study, demonstrating that the SH2 domain peptide inhibitor of GRB7 (G7-18NATE-P) completely abolished ErbB2-mediated 10A.B2 cell migration following suppression of PTP␣, stressed the important scaffolding role of GRB7. GRB7 was initially characterized as an interacting partner of ErbB2 at the tyrosine 1139 site (50) and has been implicated in the regulation of focal adhesion function and cell migration (58). Our studies also illustrate that disruption of PTP␣ expression regulates the interaction between GRB7 and ErbB2 and suggest a potential role for the GRB7 adaptor protein in the effects of PTP␣. It has been reported that GRB7 can form dimers (73), suggesting the possibility that these may provide anchorage points for proteins at focal adhesion complexes. It has been shown that phosphorylation of PTP␣ at Tyr-789, previously identified as a binding site for GRB2 (74), was critical in targeting PTP␣ to focal adhesions (75,76). It would be interesting to investigate whether GRB7 is a candidate SH2-containing protein that mediates the recruitment of PTP␣ to focal adhesions. A phosphotyrosine displacement mechanism has been proposed to facilitate the activation of SRC by PTP␣ in which pTyr-789 of PTP␣ engages the SH2 domain of SRC, thereby exposing the C-terminal pTyr for dephosphorylation and activation of the kinase (77). Perhaps pTyr-789, functioning as a GRB7 docking site on PTP␣, could provide a competing phospho site to tyrosine 1139 on ErbB2. Therefore, the transient localization of ErbB2 at ␤1-integrin complexes may be regulated in a similar manner to that observed for the activation of SRC by PTP␣, in that engagement of pTyr-789 on the phosphatase by GRB7 may expose other sites for dephosphorylation. The presence of GRB7 together with ␤1-integrins and FAK following ErbB2 activation may also be significant because it has been reported previously that the ␤1-integrin-FAK axis controls the initial proliferation of micrometastases of mammary carcinoma cells in the lung (78).
Overall, we have shown for the first time that the suppression of PTP␣ expression leads to a GRB7-dependent increase in migration of human mammary epithelial cells in response to ErbB2 activation. Our data support a novel role for PTP␣ in regulating the phosphorylation of FAK at tyrosine 407, thereby promoting its association with vinculin at ␤1-integrin focal adhesion complexes. These novel aspects of PTP␣ signaling reveal an important role of the phosphatase in the regulation of a key mediator of focal adhesions and cell migration and of ErbB2-mediated mammary cancer cell migration.