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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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Ras is required for uPA-induced MAP kinase activation and MCF-7 cell migration. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and dominant-negative H-Ras, constitutively active H-Ras, or the empty vector, pDCR. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) for 1 min. HA-tagged ERK1 was recovered by immunoprecipitation. Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified H-Ras mutants and GFP or GFP alone. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) and allowed to migrate for 6 h on serum-coated membranes. Cellular migration was standardized to that observed with mock-transfected cells that were not uPA-treated.
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Figure 4: Ras is required for uPA-induced MAP kinase activation and MCF-7 cell migration. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and dominant-negative H-Ras, constitutively active H-Ras, or the empty vector, pDCR. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) for 1 min. HA-tagged ERK1 was recovered by immunoprecipitation. Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified H-Ras mutants and GFP or GFP alone. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) and allowed to migrate for 6 h on serum-coated membranes. Cellular migration was standardized to that observed with mock-transfected cells that were not uPA-treated.

Mentions: Ras activation is frequently but not always necessary as an upstream activator of ERK in growth factor–stimulated cells (Frost et al. 1997). To test whether Ras is involved in the uPAR-initiated signaling pathway which leads to ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1 and either dominant-negative H-Ras or constitutively active H-Ras. As shown in Fig. 4 A, phosphorylated HA-ERK1 was not detected in cells that were transfected to express dominant-negative H-Ras, irrespective of whether the cells were treated with uPA or not. By contrast, cells that were transfected to express constitutively active H-Ras demonstrated substantial levels of phosphorylated HA-ERK1; however, the level of phosphorylated HA-ERK1 was not further increased by uPA. As a control, we transfected cells with the empty vector, pDCR. Trace levels of phosphorylated HA-ERK1 were detected in the absence of uPA; however, when these cells were treated with DIP-uPA, a substantial increase in phosphorylated HA-ERK1 was observed. In other control experiments, we demonstrated unchanged binding of 125I-DIP-uPA to constitutively active H-Ras–expressing MCF-7 cells, indicating that cell-surface uPAR expression is not altered in these cells (data not shown).


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)

Ras is required for uPA-induced MAP kinase activation and MCF-7 cell migration. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and dominant-negative H-Ras, constitutively active H-Ras, or the empty vector, pDCR. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) for 1 min. HA-tagged ERK1 was recovered by immunoprecipitation. Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified H-Ras mutants and GFP or GFP alone. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) and allowed to migrate for 6 h on serum-coated membranes. Cellular migration was standardized to that observed with mock-transfected cells that were not uPA-treated.
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Related In: Results  -  Collection

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Figure 4: Ras is required for uPA-induced MAP kinase activation and MCF-7 cell migration. (A) MCF-7 cells were cotransfected to express HA-tagged ERK1 and dominant-negative H-Ras, constitutively active H-Ras, or the empty vector, pDCR. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) for 1 min. HA-tagged ERK1 was recovered by immunoprecipitation. Phosphorylated and total levels of ERK1 were determined by immunoblot analysis. (B) MCF-7 cells were transfected to express the specified H-Ras mutants and GFP or GFP alone. The cells were then treated with 10 nM DIP-uPA (+) or vehicle (−) and allowed to migrate for 6 h on serum-coated membranes. Cellular migration was standardized to that observed with mock-transfected cells that were not uPA-treated.
Mentions: Ras activation is frequently but not always necessary as an upstream activator of ERK in growth factor–stimulated cells (Frost et al. 1997). To test whether Ras is involved in the uPAR-initiated signaling pathway which leads to ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1 and either dominant-negative H-Ras or constitutively active H-Ras. As shown in Fig. 4 A, phosphorylated HA-ERK1 was not detected in cells that were transfected to express dominant-negative H-Ras, irrespective of whether the cells were treated with uPA or not. By contrast, cells that were transfected to express constitutively active H-Ras demonstrated substantial levels of phosphorylated HA-ERK1; however, the level of phosphorylated HA-ERK1 was not further increased by uPA. As a control, we transfected cells with the empty vector, pDCR. Trace levels of phosphorylated HA-ERK1 were detected in the absence of uPA; however, when these cells were treated with DIP-uPA, a substantial increase in phosphorylated HA-ERK1 was observed. In other control experiments, we demonstrated unchanged binding of 125I-DIP-uPA to constitutively active H-Ras–expressing MCF-7 cells, indicating that cell-surface uPAR expression is not altered in these cells (data not shown).

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