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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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αVβ5 and a β1-containing integrin mediate MCF-7 cell migration in the presence and absence of uPA. (A) MCF-7 cells were treated with increasing concentrations of blocking antibodies against αVβ3 (LM609), αVβ5 (P1F6), and/or β1-integrin subunit (6S6) and allowed to migrate for 6 h in Transwell chambers with vitronectin-coated membranes. (B) MCF-7 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) in the presence of the specified antibodies (32 μg/ml) and allowed to migrate for 6 h on vitronectin-coated membranes. Control cells were not treated with antibodies. Migration was expressed as a percentage of that observed with control cells in the absence of DIP-uPA.
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Figure 10: αVβ5 and a β1-containing integrin mediate MCF-7 cell migration in the presence and absence of uPA. (A) MCF-7 cells were treated with increasing concentrations of blocking antibodies against αVβ3 (LM609), αVβ5 (P1F6), and/or β1-integrin subunit (6S6) and allowed to migrate for 6 h in Transwell chambers with vitronectin-coated membranes. (B) MCF-7 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) in the presence of the specified antibodies (32 μg/ml) and allowed to migrate for 6 h on vitronectin-coated membranes. Control cells were not treated with antibodies. Migration was expressed as a percentage of that observed with control cells in the absence of DIP-uPA.

Mentions: In the absence of uPA, αVβ3-blocking antibody had no effect on MCF-7 cell migration on vitronectin, as anticipated (Fig. 10 A). However, αVβ5-blocking antibody was also inactive and β1 subunit–blocking antibody inhibited migration by <25%. When added in combination, αVβ5-blocking antibody and the β1-blocking antibody inhibited migration by up to 79 ± 5%. These results suggest that αVβ5 and a β1-containing integrin (probably αVβ1) function interchangeably in MCF-7 cell migration on vitronectin, in the 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)

αVβ5 and a β1-containing integrin mediate MCF-7 cell migration in the presence and absence of uPA. (A) MCF-7 cells were treated with increasing concentrations of blocking antibodies against αVβ3 (LM609), αVβ5 (P1F6), and/or β1-integrin subunit (6S6) and allowed to migrate for 6 h in Transwell chambers with vitronectin-coated membranes. (B) MCF-7 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) in the presence of the specified antibodies (32 μg/ml) and allowed to migrate for 6 h on vitronectin-coated membranes. Control cells were not treated with antibodies. Migration was expressed as a percentage of that observed with control cells in the absence of DIP-uPA.
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Related In: Results  -  Collection

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Figure 10: αVβ5 and a β1-containing integrin mediate MCF-7 cell migration in the presence and absence of uPA. (A) MCF-7 cells were treated with increasing concentrations of blocking antibodies against αVβ3 (LM609), αVβ5 (P1F6), and/or β1-integrin subunit (6S6) and allowed to migrate for 6 h in Transwell chambers with vitronectin-coated membranes. (B) MCF-7 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) in the presence of the specified antibodies (32 μg/ml) and allowed to migrate for 6 h on vitronectin-coated membranes. Control cells were not treated with antibodies. Migration was expressed as a percentage of that observed with control cells in the absence of DIP-uPA.
Mentions: In the absence of uPA, αVβ3-blocking antibody had no effect on MCF-7 cell migration on vitronectin, as anticipated (Fig. 10 A). However, αVβ5-blocking antibody was also inactive and β1 subunit–blocking antibody inhibited migration by <25%. When added in combination, αVβ5-blocking antibody and the β1-blocking antibody inhibited migration by up to 79 ± 5%. These results suggest that αVβ5 and a β1-containing integrin (probably αVβ1) function interchangeably in MCF-7 cell migration on vitronectin, in the 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