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. 1999 Apr;19(4):3125-35.
doi: 10.1128/MCB.19.4.3125.

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility

S Mañes  1 , E Mira, C Gómez-Mouton, Z J Zhao, R A Lacalle, C Martínez-A
Affiliations

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility

S Mañes et al. Mol Cell Biol. 1999 Apr.

Abstract

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.

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Figures

FIG. 1
FIG. 1
IGF-I induces MCF-7 cell migration in the absence of de novo protein synthesis. (A) MCF-7 cells were incubated with αIR3 (0.2 μg/ml), anti-αvβ5, anti-αv, or immunoglobulin G as control (20 μg/ml in all cases) or CHx (5 μg/ml) and seeded in vitronectin-coated transwells, and IGF-I (1 ng/ml) was added to the lower chamber. The number of migrating cells represented is the mean ± standard deviation counted per filter. (B) Serum-starved MCF-7 cells were incubated with the indicated amounts of IGF-I in the presence (●) or absence (■) of CHx. [3H]TdR incorporation was determined after 24 h. The proliferation observed in the absence of IGF-I was considered 100%.
FIG. 2
FIG. 2
IGF-I induces tyrosine dephosphorylation of FAK and paxillin. Cell lysates from unstimulated (time 0) or IGF-I-stimulated MCF-7 cells seeded on vitronectin were immunoprecipitated with anti-phosphotyrosine antibody (PY) and resolved on polyacrylamide gels. The membranes were blotted with the indicated antibodies, and blotting was followed by enhanced chemiluminescence analysis. w.b., Western blot; i.p., immunoprecipitation.
FIG. 3
FIG. 3
IGF-I enhances MCF-7 adhesion to vitronectin. (A) Serum-starved MCF-7 cells were detached with a trypsin-EDTA solution and, after two washes in PBS, incubated with the indicated doses of IGF-I in DMEM-BSA for 10 min. The mixture was then added to vitronectin-coated 96-well plates and incubated for an additional 10 min. After two washes, attached cells were assessed by crystal violet staining. (B) MCF-7 cells were detached and incubated in DMEM without BSA (30 min, 37°C). IGF-I (10 ng/ml) was added, and the cells were then incubated for the indicated times under the same conditions. IGF-I was removed by pelleting the cells and washing twice with PBS. Finally, cells were resuspended in DMEM-BSA and added to the vitronectin-coated plates. After washing with PBS, the attached cells were assessed by crystal violet staining. (C) Cells prepared as just described were lysed before (nonattached) or after (attached) adhesion to vitronectin plates. Cell lysates were immunoprecipitated (i.p.) with an FAK antibody, and the resolved proteins were analyzed by Western immunoblotting (w.b.) with anti-phosphotyrosine (PY) and anti-FAK antibodies. O.D., optical density.
FIG. 4
FIG. 4
IGF-1R-associated FAK is dephosphorylated. Vitronectin-attached MCF-7 cells were stimulated with IGF-I for the times indicated (time 0 represents no stimulation) at 37°C. Cells were then washed twice in PBS to remove unbound IGF-I, and cell lysates were immunoprecipitated (i.p.) with the anti-IGF-I antibody KM5A1. The resolved proteins were Western immunoblotted (w.b.) sequentially with the indicated antibodies.
FIG. 5
FIG. 5
Characterization of SHP-2-deficient clones. (A) Western immunoblot (w.b.) analysis of ERK kinases. Equal amounts of cell lysates (30 μg) of empty vector- (mock), wild-type SHP-2 (SHP2-wt), or mutant SHP2-C>S-transfected cells, stimulated with IGF-I at the times indicated, were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-phospho-Erk-specific (upper panel) or anti-Erk antibodies (lower panel). These antibodies recognize both Erk-1 and Erk-2. (B and C) Determination of ectopically expressed SHP-2. In panel B, equal amounts of cell lysates (30 μg) were resolved by SDS-PAGE and immunoblotted with an anti-SHP-2 antibody. In panel C, total RNA was isolated from mock-, SHP2-wt-, and SHP2-C>S-transfected cells, and reverse transcriptase PCR was performed using specific primers for either SHP-2 (upper left panel) or β-actin (lower left panel). The primers used do not amplify nonreverse transcribed RNA (data not shown). Finally, a part of the PCR products was incubated with PstI (right panel), which digests the endogenous and the ectopically expressed SHP2-wt forms, but not the SHP2-C>S form. (D) Determination of integrin levels. MCF-7 cells, transfected as in panel A, were stained with an anti-αvβ5 antibody followed by a fluorescein-labelled second antibody and were then analyzed by FACS.
FIG. 6
FIG. 6
IGF-I-induced FAK dephosphorylation is dependent on SHP-2 catalytic activity. MCF-7 cells transfected with wild-type SHP-2 (SHP2-wt) or the catalytically inactive mutant SHP-2-C>S were stimulated with IGF-I at the times indicated (time 0 represents the absence of stimulus). After a washing step, cell lysates were prepared and the proteins were immunoprecipitated (i.p.) with an anti-FAK antibody and resolved by SDS-PAGE. Western immunoblotting (w.b.) was performed sequentially with anti-phosphotyrosine (PY, upper panel) and, as a protein loading control, anti-FAK (lower panel) antibodies.
FIG. 7
FIG. 7
SHP-2 catalytic activity is required for IGF-I-triggered MCF-7 invasion. (A) MCF-7 transfected with empty vector (mock), wild-type SHP-2, or the mutant SHP-2-C>S were seeded on vitronectin-coated transwells, and IGF-I (1 ng/ml) was added to the lower chamber. The number of migrating mock-transfected cells was designated 100%. Two representative SHP-2-C>S-transfected cell clones are shown (#3 and #14). (B) The SHP2-C>S–MCF-7 clone #3 cell line was untransfected (untransf.) or transfected with the empty vector (vector) or with the vector coding for wild-type SHP-2 (SHP2-wt) at the indicated doses and then assayed for IGF-I-triggered invasion through vitronectin as in panel A. The number of migrating mock-transfected cells was designated 100%. (C) SHP2-wt mRNA expression in retransfected SHP2-C>S cells. As described in the legend to Fig. 5, total RNA was amplified by reverse transcriptase PCR using specific SHP-2 primers, and PCR products were digested with PstI. The figure shows the restriction analysis for mock-transfected cells (lane 1) and for SHP2-C>S cells alone (lane 2) transfected with empty vector (lane 3) or with SHP2-wt plasmid (5 μg) (lane 4). (D) Western blot analysis of SHP-2 levels in the cells used in panel C. Equal amounts of cell lysate were resolved by SDS-PAGE and blotted with an SHP-2-specific antibody. Data represent one of three similar independent experiments.
FIG. 8
FIG. 8
SHP-2 catalytic activity regulates the number of focal adhesions. MCF-7 cells transfected with wild-type (SHP2-wt) or inactive mutant (SHP2-C>S) constructs of SHP-2 were seeded on vitronectin-coated chamber slides, fixed, permeabilized with Triton X-100, and examined for focal adhesion plaques by using a paxillin antibody followed by a Cy3-conjugated secondary antibody.
FIG. 9
FIG. 9
Effect of SHP-2 on MCF-7 cell invasion is independent of MAPK activation. (A) MCF-7 cells were incubated with the MEK inhibitor PD-98059 at the indicated concentrations for 30 min before seeding on vitronectin-coated transwells. The migration induced by IGF-I (1 ng/ml) added to the lower chamber was examined. The numbers of migrating cells are the means ± standard deviations of cells per filter. (B) Serum-starved MCF-7 cells were incubated with IGF-I (100 ng/ml) in the presence of the indicated amounts of PD-98059. [3H] TdR incorporation was determined after 24 h. The proliferation obtained in the absence of PD-98059 was considered 100%.
FIG. 10
FIG. 10
SHP-2 catalytic activity is a general requirement for cell motility. (A) MCF-7 cells transfected with empty vector (mock), wild-type SHP-2, or the mutant SHP-2-C>S were seeded on type IV collagen-coated transwells and assayed for migration in response to IGF-I (1 ng/ml). The number of migrating mock-transfected cells was designated 100%. (B) DU-145 cells transfected with empty vector (mock), wild-type SHP-2 (SHP2-wt), or inactive SHP-2-C>S (SHP2-C>S) were seeded on vitronectin-coated transwells and assayed for migration in response to IGF-I. As for panel A, the number of migrating mock-transfected cells was designated 100%.
FIG. 11
FIG. 11
Model for IGF-I-induced motility in MCF-7 cells. Schematic representation of the IGF-I-triggered signalling pathways leading to adhesion and deadhesion in MCF-7 cells. For details, see Discussion. PAX, paxillin; IRS, IRS-1; P, phosphotyrosine.
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