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Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ, Gonias SL - J. Cell Biol. (1999)

Bottom Line: When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration.Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration.Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

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MEK is required for uPA-induced MAP kinase activation and motility stimulation. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and the specified MEK mutants or the empty vector, pCHA. HA-tagged ERK1 was immunoprecipitated from cell extracts 1 min after treating the cells with 10 nM DIP-uPA (+) or with vehicle (−). Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified MEK mutants and GFP, or GFP alone (pEGFP). Mock-transfected cells were treated with Superfect in the absence of cDNAs. Cells were allowed to migrate in the presence (+) or absence (−) of 10 nM DIP-uPA for 6 h on serum-coated membranes. Cellular migration of transfected cells was determined using fluorescence microscopy to detect GFP-expressing cells. Migration of mock-transfected cells was determined by Diff-Quik staining. Mock-transfected cells migrated identically to MCF-7 cells that were not Superfect-treated, in the presence and absence of uPA (data not shown). To compare results obtained with GFP-expressing and mock-transfected cells, the number of GFP-expressing cells that penetrated the membrane was divided by the transfection efficiency. For each bar, cellular migration is expressed as a percentage of that observed with mock-transfected cells that were not treated with DIP-uPA.
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Figure 3: MEK is required for uPA-induced MAP kinase activation and motility stimulation. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and the specified MEK mutants or the empty vector, pCHA. HA-tagged ERK1 was immunoprecipitated from cell extracts 1 min after treating the cells with 10 nM DIP-uPA (+) or with vehicle (−). Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified MEK mutants and GFP, or GFP alone (pEGFP). Mock-transfected cells were treated with Superfect in the absence of cDNAs. Cells were allowed to migrate in the presence (+) or absence (−) of 10 nM DIP-uPA for 6 h on serum-coated membranes. Cellular migration of transfected cells was determined using fluorescence microscopy to detect GFP-expressing cells. Migration of mock-transfected cells was determined by Diff-Quik staining. Mock-transfected cells migrated identically to MCF-7 cells that were not Superfect-treated, in the presence and absence of uPA (data not shown). To compare results obtained with GFP-expressing and mock-transfected cells, the number of GFP-expressing cells that penetrated the membrane was divided by the transfection efficiency. For each bar, cellular migration is expressed as a percentage of that observed with mock-transfected cells that were not treated with DIP-uPA.

Mentions: To further explore the relationship between the uPA/uPAR system and ERK activation in promoting cellular migration, we transfected MCF-7 cells to express dominant-negative or constitutively active MEK1. To demonstrate that the MEK1 mutants were functional as upstream-modulators of ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1. The transfectants were treated with DIP-uPA (10 nM) or vehicle for 1 min; cell extracts were immunoprecipitated using an antibody directed against the HA-epitope and phosphorylated ERK1 was detected by immunoblot analysis. As shown in Fig. 3 A, MCF-7 cells that were transfected to express dominant-negative MEK1 did not contain detectable levels of phosphorylated HA-ERK1, irrespective of whether these cells were treated with DIP-uPA or not. By contrast, substantial levels of phosphorylated HA-ERK1 were detected in cells that were transfected to express constitutively active MEK1. However, when these cells were treated with DIP-uPA, no further increase in phosphorylated HA-ERK1 was observed. As a control, we cotransfected MCF-7 cells with the HA-tagged ERK1 construct and with the empty vector (pCHA) which had been used to prepare the constitutively active MEK1 mutant. In the absence of uPA, only trace levels of phosphorylated HA-ERK1 were observed; however, DIP-uPA substantially increased phosphorylated HA-ERK1 in these cells. These results demonstrate that the MEK1 mutants were functional as regulators of ERK activation in the presence and absence of uPA.


Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ, Gonias SL - J. Cell Biol. (1999)

MEK is required for uPA-induced MAP kinase activation and motility stimulation. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and the specified MEK mutants or the empty vector, pCHA. HA-tagged ERK1 was immunoprecipitated from cell extracts 1 min after treating the cells with 10 nM DIP-uPA (+) or with vehicle (−). Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified MEK mutants and GFP, or GFP alone (pEGFP). Mock-transfected cells were treated with Superfect in the absence of cDNAs. Cells were allowed to migrate in the presence (+) or absence (−) of 10 nM DIP-uPA for 6 h on serum-coated membranes. Cellular migration of transfected cells was determined using fluorescence microscopy to detect GFP-expressing cells. Migration of mock-transfected cells was determined by Diff-Quik staining. Mock-transfected cells migrated identically to MCF-7 cells that were not Superfect-treated, in the presence and absence of uPA (data not shown). To compare results obtained with GFP-expressing and mock-transfected cells, the number of GFP-expressing cells that penetrated the membrane was divided by the transfection efficiency. For each bar, cellular migration is expressed as a percentage of that observed with mock-transfected cells that were not treated with DIP-uPA.
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Related In: Results  -  Collection

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Figure 3: MEK is required for uPA-induced MAP kinase activation and motility stimulation. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and the specified MEK mutants or the empty vector, pCHA. HA-tagged ERK1 was immunoprecipitated from cell extracts 1 min after treating the cells with 10 nM DIP-uPA (+) or with vehicle (−). Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified MEK mutants and GFP, or GFP alone (pEGFP). Mock-transfected cells were treated with Superfect in the absence of cDNAs. Cells were allowed to migrate in the presence (+) or absence (−) of 10 nM DIP-uPA for 6 h on serum-coated membranes. Cellular migration of transfected cells was determined using fluorescence microscopy to detect GFP-expressing cells. Migration of mock-transfected cells was determined by Diff-Quik staining. Mock-transfected cells migrated identically to MCF-7 cells that were not Superfect-treated, in the presence and absence of uPA (data not shown). To compare results obtained with GFP-expressing and mock-transfected cells, the number of GFP-expressing cells that penetrated the membrane was divided by the transfection efficiency. For each bar, cellular migration is expressed as a percentage of that observed with mock-transfected cells that were not treated with DIP-uPA.
Mentions: To further explore the relationship between the uPA/uPAR system and ERK activation in promoting cellular migration, we transfected MCF-7 cells to express dominant-negative or constitutively active MEK1. To demonstrate that the MEK1 mutants were functional as upstream-modulators of ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1. The transfectants were treated with DIP-uPA (10 nM) or vehicle for 1 min; cell extracts were immunoprecipitated using an antibody directed against the HA-epitope and phosphorylated ERK1 was detected by immunoblot analysis. As shown in Fig. 3 A, MCF-7 cells that were transfected to express dominant-negative MEK1 did not contain detectable levels of phosphorylated HA-ERK1, irrespective of whether these cells were treated with DIP-uPA or not. By contrast, substantial levels of phosphorylated HA-ERK1 were detected in cells that were transfected to express constitutively active MEK1. However, when these cells were treated with DIP-uPA, no further increase in phosphorylated HA-ERK1 was observed. As a control, we cotransfected MCF-7 cells with the HA-tagged ERK1 construct and with the empty vector (pCHA) which had been used to prepare the constitutively active MEK1 mutant. In the absence of uPA, only trace levels of phosphorylated HA-ERK1 were observed; however, DIP-uPA substantially increased phosphorylated HA-ERK1 in these cells. These results demonstrate that the MEK1 mutants were functional as regulators of ERK activation in the presence and absence of uPA.

Bottom Line: When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration.Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration.Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Show MeSH
Related in: MedlinePlus