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Regulation of cell motility by mitogen-activated protein kinase.

Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA - J. Cell Biol. (1997)

Bottom Line: Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins.In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin.Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

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Stimulation of COS-7 cell migration after transfection with mutationally activated MEK1. (A, upper panel) COS-7 cells were  serum starved for 18 h and allowed to migrate for 3 h on collagen-coated membranes after transient transfection with either the empty  expression vector (pcDNA-3, control) or the expression vector containing the mutationally activated MEK1 (MEK+) as described in  Materials and Methods. In both cases, cells were cotransfected with a β-galactosidase–containing vector (pSV–β-galactosidase) for in  situ β-galactosidase staining with X-gal and a myc-tagged ERK1 reporter construct as described in Materials and Methods. This facilitated enumeration of only those migratory cells that had been positively transfected. In either case, transfection efficiency was routinely  30–40%. Alternatively, control transfectants were allowed to migrate in the presence of EGF (100 ng/ml) and then enumerated by  counting cells on the underside of the migration chamber using an Olympus (BX60) inverted microscope. Cells per high-powered (40×)  field were counted blindly by two observers. Each bar represents the mean ± SD of triplicate migration wells of an independent experiment. (Lower panel) Detergent lysates from cells treated as above were either directly immunoblotted for MEK protein expression using  rabbit anti-MEK or subjected to immunoprecipitation using anti-ERK followed by immunoblotting with antiphosphotyrosine as described in Materials and Methods. In addition, these immunoprecipitates were analyzed for MAP kinase activity using an in vitro kinase  assay and the substrate MBP as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments. (B, upper panel) COS-7 cells treated as described in A were allowed to migrate in the presence or absence of  the MEK inhibitor (25 μM, PD98059) for 2–4 h and then stained and quantitated as described above. (Lower panel) Detergent lysates  from these cells were examined for expression of MEK, ERK phosphorylation, and kinase activity using MBP as a substrate as described above.
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Figure 2: Stimulation of COS-7 cell migration after transfection with mutationally activated MEK1. (A, upper panel) COS-7 cells were serum starved for 18 h and allowed to migrate for 3 h on collagen-coated membranes after transient transfection with either the empty expression vector (pcDNA-3, control) or the expression vector containing the mutationally activated MEK1 (MEK+) as described in Materials and Methods. In both cases, cells were cotransfected with a β-galactosidase–containing vector (pSV–β-galactosidase) for in situ β-galactosidase staining with X-gal and a myc-tagged ERK1 reporter construct as described in Materials and Methods. This facilitated enumeration of only those migratory cells that had been positively transfected. In either case, transfection efficiency was routinely 30–40%. Alternatively, control transfectants were allowed to migrate in the presence of EGF (100 ng/ml) and then enumerated by counting cells on the underside of the migration chamber using an Olympus (BX60) inverted microscope. Cells per high-powered (40×) field were counted blindly by two observers. Each bar represents the mean ± SD of triplicate migration wells of an independent experiment. (Lower panel) Detergent lysates from cells treated as above were either directly immunoblotted for MEK protein expression using rabbit anti-MEK or subjected to immunoprecipitation using anti-ERK followed by immunoblotting with antiphosphotyrosine as described in Materials and Methods. In addition, these immunoprecipitates were analyzed for MAP kinase activity using an in vitro kinase assay and the substrate MBP as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments. (B, upper panel) COS-7 cells treated as described in A were allowed to migrate in the presence or absence of the MEK inhibitor (25 μM, PD98059) for 2–4 h and then stained and quantitated as described above. (Lower panel) Detergent lysates from these cells were examined for expression of MEK, ERK phosphorylation, and kinase activity using MBP as a substrate as described above.

Mentions: To further investigate the role of MAP kinase in cell motility, we used COS-7 cells since they efficiently express plasmids encoding members of the Ras/MAP kinase cascade (Howe et al., 1992; Cowley et al., 1994; Coso et al., 1995). Furthermore, COS-7 cells, like FG cells, migrate on collagen using integrin α2β1 (data not shown). In these experiments, we transiently expressed a mutationally active MEK1(MEK+) in COS-7 cells and examined their migratory properties on a collagen substrate. As shown in Fig. 2 A, upper panel, serum-starved MEK+ transfectant cells showed a four- to fivefold increase in migration relative to control cells transfected with the same expression vector without the MEK1 gene. However, the relative adhesive properties of these cells that were measured remained unchanged (data not shown). These cells also showed enhanced migration on fibronectin and vitronectin (data not shown), indicating that the mutationally active MEK1 could stimulate the general migratory properties of these cells. This induction of cell migration was associated with increased MAP kinase activity as measured by antiphosphotyrosine immunoblotting as well as increased phosphorylation of the substrate MBP (Fig. 2 A, lower panel). As expected, serum-starved cells exposed to EGF showed enhanced cell migration and increased MAP kinase activity (Fig. 2 A) (Howe et al., 1992). Importantly, cell migration induced by MEK+ expression was also blocked with the MEK inhibitor PD98059 (Fig. 2 B, upper panel), as was activation of ERK2 in these cells (Fig. 2 B, lower panel). These studies demonstrate that MAP kinase signaling events are critical for the induction of cell migration.


Regulation of cell motility by mitogen-activated protein kinase.

Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA - J. Cell Biol. (1997)

Stimulation of COS-7 cell migration after transfection with mutationally activated MEK1. (A, upper panel) COS-7 cells were  serum starved for 18 h and allowed to migrate for 3 h on collagen-coated membranes after transient transfection with either the empty  expression vector (pcDNA-3, control) or the expression vector containing the mutationally activated MEK1 (MEK+) as described in  Materials and Methods. In both cases, cells were cotransfected with a β-galactosidase–containing vector (pSV–β-galactosidase) for in  situ β-galactosidase staining with X-gal and a myc-tagged ERK1 reporter construct as described in Materials and Methods. This facilitated enumeration of only those migratory cells that had been positively transfected. In either case, transfection efficiency was routinely  30–40%. Alternatively, control transfectants were allowed to migrate in the presence of EGF (100 ng/ml) and then enumerated by  counting cells on the underside of the migration chamber using an Olympus (BX60) inverted microscope. Cells per high-powered (40×)  field were counted blindly by two observers. Each bar represents the mean ± SD of triplicate migration wells of an independent experiment. (Lower panel) Detergent lysates from cells treated as above were either directly immunoblotted for MEK protein expression using  rabbit anti-MEK or subjected to immunoprecipitation using anti-ERK followed by immunoblotting with antiphosphotyrosine as described in Materials and Methods. In addition, these immunoprecipitates were analyzed for MAP kinase activity using an in vitro kinase  assay and the substrate MBP as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments. (B, upper panel) COS-7 cells treated as described in A were allowed to migrate in the presence or absence of  the MEK inhibitor (25 μM, PD98059) for 2–4 h and then stained and quantitated as described above. (Lower panel) Detergent lysates  from these cells were examined for expression of MEK, ERK phosphorylation, and kinase activity using MBP as a substrate as described above.
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Related In: Results  -  Collection

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Figure 2: Stimulation of COS-7 cell migration after transfection with mutationally activated MEK1. (A, upper panel) COS-7 cells were serum starved for 18 h and allowed to migrate for 3 h on collagen-coated membranes after transient transfection with either the empty expression vector (pcDNA-3, control) or the expression vector containing the mutationally activated MEK1 (MEK+) as described in Materials and Methods. In both cases, cells were cotransfected with a β-galactosidase–containing vector (pSV–β-galactosidase) for in situ β-galactosidase staining with X-gal and a myc-tagged ERK1 reporter construct as described in Materials and Methods. This facilitated enumeration of only those migratory cells that had been positively transfected. In either case, transfection efficiency was routinely 30–40%. Alternatively, control transfectants were allowed to migrate in the presence of EGF (100 ng/ml) and then enumerated by counting cells on the underside of the migration chamber using an Olympus (BX60) inverted microscope. Cells per high-powered (40×) field were counted blindly by two observers. Each bar represents the mean ± SD of triplicate migration wells of an independent experiment. (Lower panel) Detergent lysates from cells treated as above were either directly immunoblotted for MEK protein expression using rabbit anti-MEK or subjected to immunoprecipitation using anti-ERK followed by immunoblotting with antiphosphotyrosine as described in Materials and Methods. In addition, these immunoprecipitates were analyzed for MAP kinase activity using an in vitro kinase assay and the substrate MBP as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments. (B, upper panel) COS-7 cells treated as described in A were allowed to migrate in the presence or absence of the MEK inhibitor (25 μM, PD98059) for 2–4 h and then stained and quantitated as described above. (Lower panel) Detergent lysates from these cells were examined for expression of MEK, ERK phosphorylation, and kinase activity using MBP as a substrate as described above.
Mentions: To further investigate the role of MAP kinase in cell motility, we used COS-7 cells since they efficiently express plasmids encoding members of the Ras/MAP kinase cascade (Howe et al., 1992; Cowley et al., 1994; Coso et al., 1995). Furthermore, COS-7 cells, like FG cells, migrate on collagen using integrin α2β1 (data not shown). In these experiments, we transiently expressed a mutationally active MEK1(MEK+) in COS-7 cells and examined their migratory properties on a collagen substrate. As shown in Fig. 2 A, upper panel, serum-starved MEK+ transfectant cells showed a four- to fivefold increase in migration relative to control cells transfected with the same expression vector without the MEK1 gene. However, the relative adhesive properties of these cells that were measured remained unchanged (data not shown). These cells also showed enhanced migration on fibronectin and vitronectin (data not shown), indicating that the mutationally active MEK1 could stimulate the general migratory properties of these cells. This induction of cell migration was associated with increased MAP kinase activity as measured by antiphosphotyrosine immunoblotting as well as increased phosphorylation of the substrate MBP (Fig. 2 A, lower panel). As expected, serum-starved cells exposed to EGF showed enhanced cell migration and increased MAP kinase activity (Fig. 2 A) (Howe et al., 1992). Importantly, cell migration induced by MEK+ expression was also blocked with the MEK inhibitor PD98059 (Fig. 2 B, upper panel), as was activation of ERK2 in these cells (Fig. 2 B, lower panel). These studies demonstrate that MAP kinase signaling events are critical for the induction of cell migration.

Bottom Line: Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins.In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin.Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

Show MeSH
Related in: MedlinePlus