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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-stimulated HT 1080 cell migration requires ERK activation and uPAR ligation. (A) HT 1080 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody (each at 25 μg/ml), or PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells that were not treated with DIP-uPA, antibodies, or inhibitors. (B) HT 1080 cells (2.5 × 105 cells) were incubated with 1 nM DIP-uPA or with vehicle for 4 h at 4°C as indicated. Some cells were pretreated with uPAR-specific antibody (25 μg/ml) for 20 min and then with DIP-uPA in the presence of antibody. HT 1080 cell detergent-extracts were subjected to SDS-PAGE on 8% slabs and transferred to nitrocellulose. Total cell-associated uPA (endogenously produced and exogenously added) was detected by immunoblot analysis, using uPA specific antibody (1:10,000), and HRP-conjugated anti–mouse IgG (Sigma, 1:10,000).
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Figure 2: uPA-stimulated HT 1080 cell migration requires ERK activation and uPAR ligation. (A) HT 1080 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody (each at 25 μg/ml), or PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells that were not treated with DIP-uPA, antibodies, or inhibitors. (B) HT 1080 cells (2.5 × 105 cells) were incubated with 1 nM DIP-uPA or with vehicle for 4 h at 4°C as indicated. Some cells were pretreated with uPAR-specific antibody (25 μg/ml) for 20 min and then with DIP-uPA in the presence of antibody. HT 1080 cell detergent-extracts were subjected to SDS-PAGE on 8% slabs and transferred to nitrocellulose. Total cell-associated uPA (endogenously produced and exogenously added) was detected by immunoblot analysis, using uPA specific antibody (1:10,000), and HRP-conjugated anti–mouse IgG (Sigma, 1:10,000).

Mentions: HT 1080 fibrosarcoma cells express uPAR (Laug et al. 1992) and demonstrate increased levels of activated ERK2 when treated with uPA (Konakova et al., 1997). In our initial experiments, we demonstrated specific binding of 125I-DIP-uPA to HT 1080 cells; the Kd was 1.2 ± 0.4 nM and the Bmax was 49 ± 4 fmol/mg of cell protein (n = 4), corresponding to ∼30,000 copies of cell-surface uPAR/cell. In Transwell assays, DIP-uPA (10 nM) increased HT 1080 cell migration 2.6 ± 0.1-fold (Fig. 2 A). The effects of DIP-uPA on HT 1080 cell migration were blocked by uPA- and uPAR-specific antibody and by PD098059. The same reagents did not affect HT 1080 cell migration in the absence of exogenously added uPA. Thus, uPAR-initiated signal transduction and ERK activation are critical for uPA-stimulated HT 1080 cell migration.


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-stimulated HT 1080 cell migration requires ERK activation and uPAR ligation. (A) HT 1080 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody (each at 25 μg/ml), or PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells that were not treated with DIP-uPA, antibodies, or inhibitors. (B) HT 1080 cells (2.5 × 105 cells) were incubated with 1 nM DIP-uPA or with vehicle for 4 h at 4°C as indicated. Some cells were pretreated with uPAR-specific antibody (25 μg/ml) for 20 min and then with DIP-uPA in the presence of antibody. HT 1080 cell detergent-extracts were subjected to SDS-PAGE on 8% slabs and transferred to nitrocellulose. Total cell-associated uPA (endogenously produced and exogenously added) was detected by immunoblot analysis, using uPA specific antibody (1:10,000), and HRP-conjugated anti–mouse IgG (Sigma, 1:10,000).
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Figure 2: uPA-stimulated HT 1080 cell migration requires ERK activation and uPAR ligation. (A) HT 1080 cells were treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody (each at 25 μg/ml), or PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells that were not treated with DIP-uPA, antibodies, or inhibitors. (B) HT 1080 cells (2.5 × 105 cells) were incubated with 1 nM DIP-uPA or with vehicle for 4 h at 4°C as indicated. Some cells were pretreated with uPAR-specific antibody (25 μg/ml) for 20 min and then with DIP-uPA in the presence of antibody. HT 1080 cell detergent-extracts were subjected to SDS-PAGE on 8% slabs and transferred to nitrocellulose. Total cell-associated uPA (endogenously produced and exogenously added) was detected by immunoblot analysis, using uPA specific antibody (1:10,000), and HRP-conjugated anti–mouse IgG (Sigma, 1:10,000).
Mentions: HT 1080 fibrosarcoma cells express uPAR (Laug et al. 1992) and demonstrate increased levels of activated ERK2 when treated with uPA (Konakova et al., 1997). In our initial experiments, we demonstrated specific binding of 125I-DIP-uPA to HT 1080 cells; the Kd was 1.2 ± 0.4 nM and the Bmax was 49 ± 4 fmol/mg of cell protein (n = 4), corresponding to ∼30,000 copies of cell-surface uPAR/cell. In Transwell assays, DIP-uPA (10 nM) increased HT 1080 cell migration 2.6 ± 0.1-fold (Fig. 2 A). The effects of DIP-uPA on HT 1080 cell migration were blocked by uPA- and uPAR-specific antibody and by PD098059. The same reagents did not affect HT 1080 cell migration in the absence of exogenously added uPA. Thus, uPAR-initiated signal transduction and ERK activation are critical for uPA-stimulated HT 1080 cell migration.

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