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Originally published In Press as doi:10.1074/jbc.M909575199 on April 20, 2000

J. Biol. Chem., Vol. 275, Issue 25, 19382-19388, June 23, 2000
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Urokinase-type Plasminogen Activator Stimulates the Ras/Extracellular Signal-regulated Kinase (ERK) Signaling Pathway and MCF-7 Cell Migration by a Mechanism That Requires Focal Adhesion Kinase, Src, and Shc

RAPID DISSOCIATION OF GRB2/SOS-SHC COMPLEX IS ASSOCIATED WITH THE TRANSIENT PHOSPHORYLATION OF ERK IN UROKINASE-TREATED CELLS*

Diem H. D. NguyenDagger §, Donna J. Webb§, Andrew D. Catling||, Qian SongDagger , Anita Dhakephalkar, Michael J. Weber||, Kodimangalam S. Ravichandran||, and Steven L. GoniasDagger **

From the Departments of Dagger  Biochemistry and Molecular Biology,  Pathology, and || Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received for publication, December 3, 1999, and in revised form, April 10, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAKY397F, kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The urokinase-type plasminogen activator receptor (uPAR)1 demonstrates diverse activities that may be important in cellular migration. By binding urokinase-type plasminogen activator (uPA), uPAR localizes proteolytic activity to the leading edge of cellular migration and thereby facilitates cellular penetration of tissue boundaries (1, 2). uPAR also binds directly to vitronectin and laterally associates with integrins within the plasma membrane, altering the strength of cellular adhesion (3-6). Furthermore, uPAR initiates cell signaling responses (7). These activities are particularly intriguing because uPAR is linked to the cell surface only by a glycosylphosphatidylinositol anchor (8). The glycosylphosphatidylinositol anchor may be critical for uPAR function, in that it affects interaction with other plasma membrane proteins and allows for proper compartmentalization of uPAR within plasma membrane microdomains (5, 9).

In MCF-7 and HT 1080 cells, binding of uPA to uPAR promotes cellular migration on vitronectin by a mechanism that requires uPAR-initiated cell signaling and activation of the Ras-dependent mitogen-activated protein kinase pathway (10, 11). The proteolytic activity of uPA is not necessary for this response because the amino-terminal fragment of uPA, which lacks the serine proteinase domains, also activates extracellular signal-regulated kinase (ERK) and promotes cellular migration (10). uPA activates ERK in other cell types as well, including endothelial cells and vascular smooth muscle cells (12, 13); however, the pathway that links uPAR to Ras remains undefined.

There is considerable evidence that uPAR-initiated signaling requires a transmembrane adaptor protein or proteins, as might be expected since uPAR is glycosylphosphatidylinositol-anchored. Integrins are strong candidates as uPAR-adaptor proteins because integrins are co-purified and co-immunoprecipitated with uPAR and co-localized with uPAR within the plasma membrane by fluorescence microscopy and resonance energy transfer (5, 14-18). Caveolin also associates with uPAR and stabilizes integrin-uPAR interactions (5, 19, 20), raising the possibility that integrins and caveolin function as a co-receptor complex for uPA-activated uPAR (20). Other potential uPAR-adaptor proteins, which may function independently of integrins, include the cytokine receptor, gp130, and G protein-coupled receptors (21-23). It has been proposed that the principal function of uPA may be to alter the conformation of uPAR so that uPAR associates with critical adaptor proteins in the plasma membrane (22-24). Consistent with this hypothesis, Fazioli et al. (22) demonstrated that peptides derived from the linker region between the first and second domains of uPAR, which contain the sequence SRSRY, activate the Src family tyrosine kinase, p56/p59hck, and demonstrate chemotactic activity, similarly to uPA.

If uPAR ligation initiates cell signaling through integrins, then similar intracellular enzymes and adaptor proteins should function in both responses. It is well established that integrin ligation causes ERK activation (25, 26). The responsible pathway may involve FAK, which binds to integrin beta -chains, undergoes autophosphorylation at Tyr-397, and recruits c-Src or other Src family tyrosine kinases (27-31). Src phosphorylates Tyr-925 in FAK, creating a binding site for Grb2/Sos complex (30, 31), which then activates Ras (32-35). c-Src and FAK may also phosphorylate Shc, which serves as an adaptor protein, recruiting Grb2 (33, 34). Furthermore, integrin clustering may cause ERK activation by FAK-independent pathways (35). For example, it has been shown that caveolin bridges integrin alpha -chains to Fyn and Shc, which results in the recruitment of Grb2 and activation of the Ras-ERK pathway (36).

The goal of the present investigation was to characterize the pathway, upstream of Ras, by which uPAR ligation leads to ERK activation and stimulation of MCF-7 cell migration. Our results demonstrate for the first time that soluble uPAR and a uPAR-derived peptide that contains the sequence SRSRY promote MCF-7 cell migration by a pathway that requires ERK activation. The uPAR-initiated pathway requires FAK, c-Src, and Shc, which have also been implicated in integrin-mediated ERK activation. The involvement of these factors suggested a role for Grb2/Sos complex. Therefore, we performed experiments demonstrating that uPA causes Sos to associate with Shc, presumably through Grb2. Association of Sos with Shc was highly transient, providing an explanation for the transient nature of ERK phosphorylation in uPA-stimulated cells.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents, Antibodies, and Expression Constructs-- Two-chain uPA was kindly provided by Drs. Jack Henkin and Andrew Mazar (Abbott Laboratories) and inactivated with 20 mM diisopropyl-fluorophosphate (Sigma) to form DIP-uPA, as described previously (10). Soluble human uPAR was from R&D Systems (Minneapolis, MN). The uPAR-derived peptide, acetyl-AVTYSRSRYLEC-amide, and the scrambled peptide, acetyl-TLVEYYSRASCR-amide, were synthesized by the University of Virginia Biomolecular Core Research Laboratory and purified by reversed-phase high pressure liquid chromatography using a Phenomenex Jupiter C18 column. Accuracy and homogeneity of the peptides was confirmed by matrix-assisted laser desorption ionization/time of flight mass spectrometry.

Polyclonal antibodies that recognize phosphorylated and total ERK were from Promega and Zymed Laboratories Inc., respectively. Shc-specific polyclonal antibody was from Transduction Laboratories (Lexington, KY). Sos1-specific polyclonal antibody C-23 was from Santa Cruz Biotechnology. Monoclonal antibody 4G10, which is specific for phosphotyrosine, was from Upstate Biotechnology Laboratories (Lake Placid, NY). Leupeptin was from Roche Molecular Biochemicals. Monoclonal anti-hemagglutinin antibody 12CA5 was from Babco. Aprotinin, phenylmethylsulfonyl fluoride, benzamidine, NaF, sodium orthovanadate, and G418 were from Sigma. The selective MEK inhibitor PD098059 was from Calbiochem.

Expression constructs that encode wild-type FAK, FAKY397F, and FAK-related non-kinase (FRNK) in pCMV were kindly provided by Dr. J. Thomas Parsons (University of Virginia). FAKY397F does not autophosphorylate and thus cannot recruit other proteins, such as Src family kinases (27) or phosphatidylinositol 3-kinase (38). FRNK is an autonomously expressed segment of the FAK gene that lacks catalytic activity. FRNK localizes to focal adhesions and inhibits FAK phosphorylation (35, 39). Expression constructs encoding wild-type c-Src and kinase-defective c-Src (kd-Src) in pcDNA3 were kindly provided by Drs. David Tice and Sarah Parsons (University of Virginia). kd-Src is mutated at residue 430 (Ala right-arrow Val) and inhibits the phosphorylation of c-Src substrates when overexpressed in cultured cells (40). Expression constructs that encode wild-type Shc and Shc that is mutated at residues 239, 240, and 317 (Tyr right-arrow Phe, Shc FFF) were in pEBG. Tyr-239, Tyr-240, and Tyr-317 are essential for Shc binding to Grb2 (41). Expression constructs for constitutively active MEK1 and HA-tagged ERK1 are previously described (11).

Cell Culture and Transfection Methods-- Low passage MCF-7 cells were kindly provided by Dr. Richard Santen (University of Virginia) and cultured in RPMI medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone), penicillin (100 units/ml), and streptomycin (100 µg/ml). These cells express about 4000 copies of uPAR and very low levels of endogenous uPA (10, 11). Cells were passaged with enzyme-free cell dissociation buffer (Life Technologies, Inc.) and maintained in culture at 37 °C for 48 h before the experiments were conducted.

ERK Phosphorylation Assays-- To detect ERK activation in MCF-7 cells that were transiently transfected to express wild-type or mutant forms of FAK, c-Src, or Shc (10 µg of each construct), cells were co-transfected to express HA-tagged ERK1 (2.5 µg). In some assays, the cells were also transfected to express constitutively active MEK1 (10 µg). Cells were maintained in culture for 36 h to optimize transient expression, serum-starved for 6 h, and then treated with DIP-uPA (10 nM) or vehicle for 1 min. Cell extracts were prepared in RIPA buffer, as described previously (11). HA-ERK1 was then immunoprecipitated using antibody 12CA5. Phosphorylated and total ERK were detected by immunoblot analysis (10). Transfection and co-transfection efficiencies were determined as described previously (11). Because co-transfection efficiencies were greater than 95%, HA-ERK1 was selectively recovered from cells that also expressed the wild-type or mutant forms of FAK, c-Src, or Shc.

Shc Phosphorylation in uPA-treated Cells-- Shc phosphorylation was studied in untransfected MCF-7 cells and in cells that were transiently transfected to overexpress HA-tagged wild-type Shc. Untransfected cells were cultured until 80% confluent and then incubated in serum-free RPMI medium for 12 h. The cells were treated with 10 nM DIP-uPA at 37 °C for the indicated times. Transfected cells were cultured for 36 h, serum-starved for 12 h, and then treated with DIP-uPA. Reactions were terminated by aspirating the medium and washing the cells with ice-cold phosphate-buffered saline, containing 1 mg/ml sodium orthovanadate. Cell extracts were prepared in 100 mM N-octyl glucoside, 10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 µg/ml E-64, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mg/ml sodium orthovanadate, and 0.4 mg/ml NaF, pH 7.4, at 4 °C. The protein content of each cell extract was measured. Shc was isolated from equal amounts of cellular protein by immunoprecipitation with Shc-specific polyclonal antibody (10 µg/ml) in 0.1% (w/v) ovalbumin, 10 mM EDTA, and protein A-agarose (Sigma) or, for transfected cells, with antibody 12CA5. Isolates were subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose. Phosphorylated Shc was detected by immunoblot analysis using phosphotyrosine-specific antibody (1 µg/ml). Total Shc antigen was detected with Shc-specific antibody (1:5000 dilution).

Co-immunoprecipitation of Sos with Shc-- MCF-7 cells were cultured until 80% confluent and incubated in serum-free RPMI for 12 h. The cells were then treated with 10 nM DIP-uPA. At the specified times, the cells were washed with ice-cold phosphate-buffered saline containing 1 mg/ml sodium orthovanadate and extracted in N-octyl glucoside. Shc was isolated by immunoprecipitation from equal amounts of cellular protein. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and electrotransferred to nitrocellulose. Sos1 was detected by immunoblot analysis using antibody C-23.

Cellular Migration Assays-- Transwell migration assays were performed as described previously (11). In brief, Costar Transwell membranes were coated on the underside surface with serum. We previously demonstrated that MCF-7 cells migrate equivalently on vitronectin and serum-coated membranes (10, 11), as anticipated because vitronectin serves as the major cell attachment and spreading factor in serum (42). MCF-7 cells (105 in 100 µl) were added to the top chamber of each Transwell unit and allowed to migrate for 6 h. Soluble uPAR or uPAR-derived synthetic peptides were added to both chambers. Fetal bovine serum (10%) was added to the bottom chamber. When specified, cells were preincubated with 50 µM PD098059 for 15 min. PD098059 was included in the top chamber at the same concentration. Migration was allowed to proceed for 6 h at 37 °C. Cellular penetration to the underside of a membrane was quantitated by counting Diff-Quik-stained cells, as described previously (10, 11).

To study the function of FAK, c-Src, and Shc in uPA-stimulated MCF-7 cell migration, cells were transfected with pEGFP (2.5 µg), which encodes green fluorescent protein (GFP), and with constructs that encode wild-type or mutant forms of each signaling protein (10 µg), using 50 µl of Superfect. In some experiments, cells were co-transfected with 10 µg of the construct that encodes constitutively active MEK1. Transfection efficiencies were typically 30-35%; however, co-transfection efficiencies were always greater than 95%, allowing us to quantitate migration of transiently transfected cells, in unselected populations, by counting green-fluorescing cells. Migration assays were performed 36 h after transfection using Biocoat cell culture inserts (Becton Dickinson). In each study, the number of migrating cells was divided by the transfection efficiency (typically 0.30-0.35) to allow direct comparison of results from separate experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A uPAR-derived Peptide Promotes MCF-7 Cell Migration in a MEK-dependent Manner-- Chymotrypsin-cleaved soluble uPARand a synthetic peptide corresponding to amino acids 84-95 (AVTYSRSRYLEC) of human uPAR activate p56/p59hck and demonstrate chemotactic activity toward a number of cell types, including THP-1 and rat vascular smooth muscle cells (22, 23). Apparently these reagents bind directly to a uPAR-adaptor protein and initiate a cellular response without the requirement for associated uPA. To determine whether MCF-7 cell migration is stimulated through a similar pathway, the activities of soluble uPAR and AVTYSRSRYLEC were assessed in Transwell migration assays. Fig. 1A shows that soluble uPAR stimulated MCF-7 cell migration and that the response was inhibited by PD098059, implicating ERK activation. The effects of soluble uPAR and those of PD098059, in the presence of soluble uPAR, were statistically significant (p < 0.01 and <0.05, respectively). The magnitude of the response to soluble uPAR was only slightly lower than that observed with DIP-uPA (10, 11).


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  		Fig. 1.   Soluble uPAR and a uPAR-derived peptide promote MCF-7 cell migration in the absence of uPA. MCF-7 cells were allowed to migrate in the presence of 10 nM soluble human uPAR (SuPAR) (A) or the indicated concentration of the peptide AVTYSRSRYLRC (B). PD098059 (50 µM) was added, as indicated by the plus sign. Control cells migrated in the absence of soluble human uPAR or peptide.

Fig. 1B shows that SRSRY-peptide promoted MCF-7 cell migration at concentrations from 0.1-100 pM (p < 0.05 for cells stimulated with 0.1 nM SRSRY-peptide compared with control cells). In each case, PD098059 blocked the increase in migration, implicating ERK. Interestingly, the response to SRSRY-peptide was biphasic; as the peptide concentration was increased above 10 pM, a decrease in response was observed. The reason for this is unclear; however, the equivalent pattern of response was observed previously, by other investigators, in p56/p59hck activation and chemotaxis experiments (22, 23). The scrambled peptide, TLVEYYSRASCR, did not affect MCF-7 cell migration (results not shown). Thus, soluble uPAR and SRSRY-peptide promote MCF-7 cell migration by an ERK-dependent pathway.

The Role of FAK in uPA-induced ERK Activation-- The requirement for ERK, in uPA-stimulated MCF-7 cell migration, has been demonstrated in experiments with PD098059 and mutant forms of MEK1 (10, 11). Ras is also required (11). In endothelial cells and in uPAR-expressing LNCaP cells, uPA promotes FAK phosphorylation; however, the significance of this response remains unclear (13, 18). To determine whether uPA-induced ERK phosphorylation requires FAK, MCF-7 cells were transfected to express wild-type FAK, FAKY397F, or FRNK. The same cells were co-transfected to express HA-ERK1, so that ERK phosphorylation could be selectively analyzed in transfected cells. In the absence of uPA, phosphorylated HA-ERK1 was not detected in cells that expressed any of the FAK variants (Fig. 2A). Similarly, phosphorylated HA-ERK1 was not detected in MCF-7 cells that were transfected to express HA-ERK1 alone (results not shown; see Ref. 11).


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  		Fig. 2.   FAK and c-SRC function in uPA-promoted ERK phosphorylation. MCF-7 cells were transfected to express HA-ERK1 and, in A, wild-type (wt) FAK, FAKY397F, or FRNK, and in B, wild-type c-Src or kd-Src. Cells that were co-transfected to express constitutively active MEK1 are labeled +. After serum starvation for 6 h, cells were treated with 10 nM DIP-uPA (+) or with vehicle (-) for 1 min. HA-ERK1 was then immunoprecipitated and subjected to immunoblot analysis using antibody that detects phosphorylated ERK and total ERK.

When treated with DIP-uPA, wild-type FAK-expressing cells demonstrated increased HA-ERK1 phosphorylation. By contrast, FAKY397F and FRNK completely blocked the uPA response. Substantial HA-ERK1 phosphorylation was observed in MCF-7 cells that were co-transfected to express FRNK and constitutively active MEK1; however, these cells did not demonstrate a further increase in phosphorylated HA-ERK1 when treated with DIP-uPA. In control experiments, we demonstrated that HA-ERK1 recovery from the various transfected cell populations was similar. These results suggest that FAK is necessary in the pathway by which uPAR-ligation leads to ERK activation.

The Function of c-Src in uPA-induced ERK Phosphorylation-- Phosphorylated HA-ERK1 was undetectable in MCF-7 cells that were transfected to express wild-type c-Src; however, when these cells were treated with DIP-uPA, substantial HA-ERK1 phosphorylation was observed (Fig. 2B). By contrast, kd-Src-expressing cells failed to respond to uPA. Cells that were co-transfected to express kd-Src and constitutively active MEK1 demonstrated HA-ERK1 phosphorylation in the absence of uPA. The extent of HA-ERK1 phosphorylation was not further increased when these cells were treated with DIP-uPA. In control experiments, nearly equivalent amounts of total HA-ERK1 were recovered from each transfected cell population. These results suggest that uPA-induced ERK phosphorylation requires enzymatically active c-Src. However, these results may also be explained if kd-Src competes with another cell signaling protein for a common docking site, such as phosphorylated Tyr-397 in FAK.

Shc Is Phosphorylated in uPA-stimulated MCF-7 Cells-- Shc functions as a central adaptor protein in a number of recently described pathways by which integrin ligation leads to ERK activation (33, 34, 36). Therefore, studies were undertaken to determine the role of Shc in uPAR signaling. Tyrosine phosphorylation of Shc is required for recruitment of Grb2/Sos complex (34, 36). When MCF-7 cells were treated with DIP-uPA, the p52 and p46 isoforms of Shc were tyrosine-phosphorylated (Fig. 3A). The level of Shc phosphorylation was significantly increased within 1 min of adding DIP-uPA (2.1 ± 0.4-fold, n = 3). Maximum levels of phosphorylated Shc were detected by 2.5 min (3.1 ± 0.2-fold, n = 3). In separate experiments, we overexpressed HA-tagged Shc in MCF-7 cells and then treated these cells with DIP-uPA. Again, we observed rapid Shc tyrosine phosphorylation (Fig. 3B). The response was near-maximal within 1 min and was sustained throughout the 30 min experiment. Rapid Shc phosphorylation is consistent with the function of this protein upstream of ERK in the uPA-initiated signaling cascade, but this does not explain the highly transient nature of ERK phosphorylation (less than 5 min) in these cells (10, 11).


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  		Fig. 3.   Shc phosphorylation in uPA-treated cells. MCF-7 cells (A) and cells that were transfected to express HA-Shc (B) were cultured in serum-free medium for 12 h and then exposed to 10 nM DIP-uPA for the specified times (0.5-30 min). Immunoprecipitation was performed with Shc-specific antibody or with nonimmune IgG in A and with antibody 12CA5 in B. Immunoblot analysis was performed using phosphotyrosine-specific antibody (p-Tyr) and Shc-specific antibody (total Shc). Control cells (lane C) were not treated with uPA.

The next series of experiments was performed to determine whether Shc is required for uPA-induced ERK activation. MCF-7 cells were transfected to express wild-type Shc or Shc FFF that cannot recruit Grb2 (41). As shown in Fig. 4, DIP-uPA increased HA-ERK1 phosphorylation in wild-type Shc-expressing cells. An equivalent increase in HA-ERK1 phosphorylation was observed in control cells that were transfected with the empty vector used to prepare Shc FFF. By contrast, uPA-induced HA-ERK1 phosphorylation was blocked in Shc FFF-expressing cells. In control experiments, we demonstrated equivalent recovery of HA-ERK1 in the various immunoprecipitates.


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  		Fig. 4.   Shc function in uPA-promoted ERK activation. MCF-7 cells were transfected to express HA-ERK1 and wild-type (wt) Shc or Shc FFF. Other cells were transfected with the empty vector (pEBG). Cells that were co-transfected to express constitutively active MEK1 are labeled +. After serum starvation, cells were treated with 10 nM DIP-uPA (+) or with vehicle (-) for 1 min. HA-ERK1 was then immunoprecipitated and subjected to immunoblot analysis using antibody that detects phosphorylated ERK and total ERK.

The Function of FAK, c-Src, and Shc in uPA-stimulated MCF-7 Cell Migration-- uPA-stimulated MCF-7 cell migration requires Ras and ERK (10, 11). Because our results demonstrated that dominant-negative forms of FAK, c-Src, and Shc block ERK phosphorylation, studies were undertaken to determine whether the same mutants also inhibit uPA-stimulated MCF-7 cell migration. Assays were performed using membranes that were serum-coated only on the lower surface (10). GFP expression does not affect MCF-7 cell migration or adhesion (11).

MCF-7 cells that were transfected to express GFP alone demonstrated a 2.5 ± 0.2-fold increase in cellular migration when treated with 10 nM DIP-uPA (Fig. 5A), consistent with our previous observations (11). In the absence of uPA, overexpression of wild-type FAK caused a small but significant increase in cellular migration (1.6 ± 0.1-fold, p < 0.05), as has been previously reported in other cell types (43). A further increase in cellular migration was observed when these cells were treated with 10 nM DIP-uPA (2.1-fold, p < 0.05). Thus, overexpression of wild-type FAK does not preclude response to uPA.


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  		Fig. 5.   Dominant-negative variants of FAK, c-Src, and Shc inhibit MCF-7 cell migration. MCF-7 cells were transfected to express GFP alone (pEGFP) or GFP together with the specified signaling proteins. After 36 h, the cells were transferred to Biocoat cell culture inserts and allowed to migrate across serum-coated membranes for 6 h. Migration occurred in the presence of 10 nM DIP-uPA (+) or vehicle (-). Migration was expressed as a percentage of that observed with control cells that expressed GFP alone and that were not treated with DIP-uPA (n = 4, mean ± S.E.).

FAKY397F and FRNK did not significantly alter basal MCF-7 cell migration; however, both FAK variants completely blocked the MCF-7 cell response to DIP-uPA. Similar results were obtained in experiments with mutant forms of c-Src and Shc. As shown in Fig. 5B, wild-type c-Src increased MCF-7 cell migration 1.4 ± 0.2-fold (p = 0.05) but did not inhibit the effects of DIP-uPA. By contrast, kd-Src had no effect on basal MCF-7 cell migration, but it blocked the response to uPA. Fig. 5C shows that cells that express wild-type Shc or Shc FFF migrate equivalently to the control cells. DIP-uPA stimulated wild-type Shc-expressing cells to migrate (p < 0.05) but failed to stimulate Shc FFF-expressing cells.

In control experiments, we examined the rate of adhesion of each transfected cell population to serum-coated cell culture wells. Adherent GFP-expressing cells were selectively detected by measuring surface-associated fluorescence in a Cytofluor 2350, as described previously (11). GFP fluorescence intensity is not altered by cell spreading. Cells that were transfected to express wild-type FAK, FAKY397F, FRNK, c-Src, kd-Src, wild-type Shc, or Shc FFF adhered at an equivalent rate (results not shown). These results suggest that the mutant signaling proteins regulate MCF-7 cell migration by affecting the rate of membrane penetration toward the bottom Transwell chamber after the cells have adhered to the surface.

Rescue of MCF-7 Cell Migration with Constitutively Active MEK1-- We previously demonstrated that MEK is necessary for uPA-promoted MCF-7 cell migration and that expression of constitutively active MEK1 increases cellular migration comparably to uPA (11). Because the mutant forms of FAK, c-Src, and Shc blocked uPA-promoted MCF-7 cell migration and ERK phosphorylation, studies were performed to determine whether constitutively active MEK1 restores the migratory phenotype. As shown in Fig. 6A, FRNK-expressing cells migrated at an increased rate when co-transfected to express constitutively active MEK1. The increase in migration was equivalent to that observed when control cells were treated with uPA. No further increase in cellular migration was observed when FRNK/MEK1-expressing cells were treated with DIP-uPA.


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  		Fig. 6.   Effects of constitutively active MEK1 on MCF-7 cell migration. MCF-7 cells were transfected to express GFP alone (pEGFP) or GFP and FRNK (A), kd-Src (B), or Shc FFF (C). Some cells were also transfected to express constitutively active MEK1 (+). After 36 h, cells were allowed to migrate in the presence or absence of 10 nM DIP-uPA for 6 h. Migration was expressed as a percentage of that observed with control cells that expressed GFP alone and that were not treated with DIP-uPA (n = 4, mean ± S.E.).

Cells that expressed kd-Src and Shc FFF also migrated at an increased rate when transfected to express constitutively active MEK1 (Fig. 6, B and C). In each case, mutant MEK1 increased cellular migration to the same extent as was observed when control cells were treated with DIP-uPA (labeled pEGFP in the figure) or when constitutively active MEK1 was expressed in the absence of other mutant signaling proteins (11). These results suggest that MCF-7 cell migration occurs independently of FAK, Src, and Shc when MEK1 is constitutively active.

A Mechanism for the Transient Activation of ERK in uPA-treated MCF-7 Cells-- ERK is rapidly phosphorylated in uPA-treated MCF-7 cells; however, the level of ERK phosphorylation returns to baseline by 5 min (10, 11). A number of negative feedback loops exist in the Ras-ERK pathway. These pathways may limit further ERK phosphorylation so that the activity of mitogen-activated protein kinase phosphatases predominate. Previously described negative feedback loops include MEK-dependent Sos phosphorylation, which promotes Sos dissociation from Grb2 and Shc (44), and MEK-dependent Raf-1 phosphorylation, which promotes Raf-1 relocalization away from the plasma membrane (45). Because Shc phosphorylation was sustained in uPA-treated cells, studies were undertaken to determine whether Sos remains associated with Shc. As shown in Fig. 7, Sos was recovered in Shc immunoprecipitates 1 and 2.5 min after treating MCF-7 cells with DIP-uPA but not in control cells. However, when the cells were treated with uPA for longer periods of time, association of Sos with Shc was no longer demonstrated. The transient association of Sos with Shc signaling complexes, in uPA-treated MCF-7 cells, provides a mechanistic explanation for the transient phosphorylation of ERK.


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  		Fig. 7.   Sos co-immunoprecipitation with Shc in uPA-treated cells. MCF-7 cells were treated with DIP-uPA for the indicated times. Shc was isolated by immunoprecipitation. The immunoprecipitates were then subjected to immunoblot analysis with Sos1-specific antibody. Control cells were not treated with uPA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cellular migration requires orchestration of diverse biochemical processes that may be controlled by different cell signaling pathways (46). In MCF-7 and HT 1080 cells, uPA promotes cellular migration by activating the Ras-ERK pathway (10, 11). The same pathway is also important in promoting cellular migration in response to growth factors and integrin ligation, based on its ability to alter integrin activation states (47), promote focal adhesion disassembly (48), and activate myosin light chain kinase (11, 49, 50). These activities may be most important for uropod retraction. Similar activities have been attributed to signaling proteins that function upstream of ERK. For example, c-Src weakens interactions between alpha Vbeta 3 and the cytoskeleton in focal adhesions (51).

We previously demonstrated that uPA binding to uPAR is required for ERK activation and stimulation of migration (11); however, the factors that link uPAR to Ras were undetermined. Addressing this problem was not straightforward because the nature of the adaptor proteins that associate with uPAR to transduce signaling responses remains unresolved. There is substantial evidence that integrins may function in this capacity; however, gp130 and/or G protein-coupled receptors may also be involved (21-23). Each putative adaptor protein may be coupled to different signaling effectors that could serve to link uPAR to Ras. To determine the function of FAK, c-Src, and Shc in uPA signaling, we transfected MCF-7 cells to express derivatives of these signaling proteins with dominant-negative activity. In each case, we analyzed ERK phosphorylation and cellular migration on serum-coated surfaces. FAK phosphorylation in response to uPA has been demonstrated previously (13, 18); however, our results demonstrate for the first time that FAK is necessary for uPA-stimulated ERK phosphorylation and cellular migration.

As with FAK, the role of c-Src in uPA signaling has not been clearly defined. In smooth muscle cells, uPA induces the subcellular relocalization of c-Src to the plasma membrane, preferentially toward the leading-edge of migration (23). uPA also causes cytoskeletal reorganization in c-Src+/+ but not c-Src-/- fibroblasts (23). However, in endothelial cells, uPA activates ERK by a pathway that is not affected by a general antagonist of Src family kinases (13). Our results with kd-Src support a model in which c-Src is necessary for uPA-induced ERK activation and MCF-7 cell migration; however, we cannot rule out the possibility that kd-Src functions by competing with a different enzyme or adaptor protein for a common docking site, such as phosphorylated FAK-397.

Shc was phosphorylated within 1 min in uPA-treated MCF-7 cells. Because tyrosine-phosphorylated Shc recruits Grb2/Sos complex (34, 36, 41), our results suggested that Shc may play a critical role in coupling uPAR to Ras. To test this hypothesis, we transfected MCF-7 cells to express Shc FFF and demonstrated that these cells do not respond to uPA in ERK phosphorylation assays and in cellular migration experiments. Thus, we propose that ERK is activated by a pathway that requires FAK, Src family kinases, Shc, and Ras. Although we cannot precisely define the relationship of these factors in activating the Ras-ERK pathway, it is significant that the uPAR-initiated pathway shares many similarities with pathways that link integrins to ERK (29-33).

Shc phosphorylation was sustained after ERK dephosphorylation was complete in uPA-treated MCF-7 cells. Various feedback inhibition pathways may account for this. For example, Raf may be hyperphosphorylated by enzymes downstream of MEK in the Ras-ERK pathway, causing a redistribution of Raf away from the plasma membrane (45). ERK or another enzyme downstream of MEK may also phosphorylate Sos, promoting Sos dissociation from Grb2 and Shc (44). In uPA-treated MCF-7 cells, Sos rapidly dissociated from Shc despite sustained Shc phosphorylation. Thus, integrity of the Shc-Grb2/Sos complex may be a major factor in determining the transient nature of ERK phosphorylation in uPA-treated cells. We propose a model in which 10 nM DIP-uPA rapidly saturates cell-surface uPAR. FAK, Src family kinases, and Shc are recruited to the plasma membrane to form signaling complexes. Phosphorylated Shc then recruits Grb2/Sos complex. Because DIP-uPA does not react with plasminogen activator-inhibitor-1, DIP-uPA-uPAR complex is stable on the cell surface, and free uPAR is not regenerated by endocytosis and recycling (52-55). In this scenario, Sos dissociation from Grb2 and Shc may leave residual upstream signaling complexes incapable of activating Ras. Without this linkage, the activity of mitogen-activated protein kinase phosphatases dominate and ERK dephosphorylation is observed (56, 57).

Extremely low levels of uPAR occupancy may be sufficient to promote ERK phosphorylation (10, 11, 55). In fact, uPA that is produced endogenously by cells in culture activates uPAR, via an autocrine pathway, and thereby serves as a major determinant of the basal level of ERK phosphorylation (37, 55, 58). In addition to its effects on cellular migration, ERK also regulates the transition between tumor cell dormancy and proliferation (58). Which ERK activity is observed in a specific uPA-treated cell type may depend on diverse cellular properties, including the kinetics of ERK activation/deactivation. Understanding this process in different cell types represents a challenge for future study.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL60551 and by the Susan G. Komen Breast Cancer Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Depts. of Pathology and Biochemistry and Molecular Genetics, University of Virginia, Box 800214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-982-0283; E-mail: slg2t@virginia.edu.

Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M909575199

    ABBREVIATIONS

The abbreviations used are: uPAR, urokinase-type plasminogen activator receptor; uPA, urokinase-type plasminogen activator; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; DIP, diisopropyl phospho; FRNK, FAK-related non-kinase; kd-Src, kinase-defective c-Src; MEK, mitogen-activated protein kinase/ERK kinase; GFP, green fluorescent protein; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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