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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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uPA-promoted cellular migration is matrix protein–selective. Transwell membranes were coated with purified vitronectin or with type I collagen. MCF-7 cells that were treated with mannosamine (+) and untreated cells (−) were allowed to migrate for 6 h in the presence of 10 nM DIP-uPA (+) or vehicle (−). Migration was expressed as a percentage of that observed with control cells (no mannosamine or DIP-uPA treatment) on vitronectin-coated membranes.
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Figure 8: uPA-promoted cellular migration is matrix protein–selective. Transwell membranes were coated with purified vitronectin or with type I collagen. MCF-7 cells that were treated with mannosamine (+) and untreated cells (−) were allowed to migrate for 6 h in the presence of 10 nM DIP-uPA (+) or vehicle (−). Migration was expressed as a percentage of that observed with control cells (no mannosamine or DIP-uPA treatment) on vitronectin-coated membranes.

Mentions: When surfaces are coated with serum, vitronectin serves as the major attachment and spreading factor (Hayman et al. 1985). To determine whether uPA-promoted MCF-7 cell migration is matrix protein–selective, Transwell membranes were coated with purified vitronectin or type I collagen, instead of serum. DIP-uPA increased MCF-7 cell migration on vitronectin 3.1 ± 0.2-fold , as anticipated (Fig. 8). MCF-7 cells migrated more rapidly on type I collagen-coated surfaces, in the absence of uPA; however, DIP-uPA failed to stimulate cellular migration further. In control experiments, we demonstrated that MCF-7 cell migration across collagen-coated membranes occurs as a linear function of time (1–6 h), precluding the possibility that we missed a uPA response due to the assay time (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)

uPA-promoted cellular migration is matrix protein–selective. Transwell membranes were coated with purified vitronectin or with type I collagen. MCF-7 cells that were treated with mannosamine (+) and untreated cells (−) were allowed to migrate for 6 h in the presence of 10 nM DIP-uPA (+) or vehicle (−). Migration was expressed as a percentage of that observed with control cells (no mannosamine or DIP-uPA treatment) on vitronectin-coated membranes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199739&req=5

Figure 8: uPA-promoted cellular migration is matrix protein–selective. Transwell membranes were coated with purified vitronectin or with type I collagen. MCF-7 cells that were treated with mannosamine (+) and untreated cells (−) were allowed to migrate for 6 h in the presence of 10 nM DIP-uPA (+) or vehicle (−). Migration was expressed as a percentage of that observed with control cells (no mannosamine or DIP-uPA treatment) on vitronectin-coated membranes.
Mentions: When surfaces are coated with serum, vitronectin serves as the major attachment and spreading factor (Hayman et al. 1985). To determine whether uPA-promoted MCF-7 cell migration is matrix protein–selective, Transwell membranes were coated with purified vitronectin or type I collagen, instead of serum. DIP-uPA increased MCF-7 cell migration on vitronectin 3.1 ± 0.2-fold , as anticipated (Fig. 8). MCF-7 cells migrated more rapidly on type I collagen-coated surfaces, in the absence of uPA; however, DIP-uPA failed to stimulate cellular migration further. In control experiments, we demonstrated that MCF-7 cell migration across collagen-coated membranes occurs as a linear function of time (1–6 h), precluding the possibility that we missed a uPA response due to the assay time (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