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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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MAP kinase activity  is associated with increased  MLC phosphorylation. (A)  COS-7 cells transfected with  empty vector or MEK+ together with HA-tagged  ERK2 reporter construct  were labeled metabolically  for 2 h with [32P]orthophosphate in the presence or absence of the MEK inhibitor  (25 μM). Cells were then  lysed in detergent and analyzed (20 μg total cell protein) by SDS-PAGE and  autoradiography for changes  in phosphorylation as described in Materials and  Methods. Arrows depict expected migration of HAERK2 and MLC. (B) MLC or  HA-tagged ERK2 were immunoprecipitated from the above lysates with anti–myosin IIB or anti-HA specific antibodies, respectively, and analyzed for changes in  phosphorylation as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments.
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Figure 3: MAP kinase activity is associated with increased MLC phosphorylation. (A) COS-7 cells transfected with empty vector or MEK+ together with HA-tagged ERK2 reporter construct were labeled metabolically for 2 h with [32P]orthophosphate in the presence or absence of the MEK inhibitor (25 μM). Cells were then lysed in detergent and analyzed (20 μg total cell protein) by SDS-PAGE and autoradiography for changes in phosphorylation as described in Materials and Methods. Arrows depict expected migration of HAERK2 and MLC. (B) MLC or HA-tagged ERK2 were immunoprecipitated from the above lysates with anti–myosin IIB or anti-HA specific antibodies, respectively, and analyzed for changes in phosphorylation as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments.

Mentions: Potential mediators of cell migration downstream of MAP kinase were identified by examining the phosphoprotein profile from MEK+ COS-7 cells metabolically labeled with [32P]orthophosphate. We observed in MEK+ but not control transfectants the presence of labeled proteins migrating at ∼20 kD that comigrated with MLC (Fig. 3 A). We then subjected these same lysates to immunoprecipitation using an antibody directed to myosin IIB since this form of myosin is present in COS cells (Klemke, R., unpublished data). This antibody precipitated the 20-kD light chains from these cells, which showed increased phosphate incorporation in the MEK+ cells relative to vector transfected control cells Fig. 3 B. In addition, the phosphorylation of this protein was blocked by pretreatment of MEK+ cells with the MEK inhibitor PD98059 (Fig. 3).


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)

MAP kinase activity  is associated with increased  MLC phosphorylation. (A)  COS-7 cells transfected with  empty vector or MEK+ together with HA-tagged  ERK2 reporter construct  were labeled metabolically  for 2 h with [32P]orthophosphate in the presence or absence of the MEK inhibitor  (25 μM). Cells were then  lysed in detergent and analyzed (20 μg total cell protein) by SDS-PAGE and  autoradiography for changes  in phosphorylation as described in Materials and  Methods. Arrows depict expected migration of HAERK2 and MLC. (B) MLC or  HA-tagged ERK2 were immunoprecipitated from the above lysates with anti–myosin IIB or anti-HA specific antibodies, respectively, and analyzed for changes in  phosphorylation as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139771&req=5

Figure 3: MAP kinase activity is associated with increased MLC phosphorylation. (A) COS-7 cells transfected with empty vector or MEK+ together with HA-tagged ERK2 reporter construct were labeled metabolically for 2 h with [32P]orthophosphate in the presence or absence of the MEK inhibitor (25 μM). Cells were then lysed in detergent and analyzed (20 μg total cell protein) by SDS-PAGE and autoradiography for changes in phosphorylation as described in Materials and Methods. Arrows depict expected migration of HAERK2 and MLC. (B) MLC or HA-tagged ERK2 were immunoprecipitated from the above lysates with anti–myosin IIB or anti-HA specific antibodies, respectively, and analyzed for changes in phosphorylation as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments.
Mentions: Potential mediators of cell migration downstream of MAP kinase were identified by examining the phosphoprotein profile from MEK+ COS-7 cells metabolically labeled with [32P]orthophosphate. We observed in MEK+ but not control transfectants the presence of labeled proteins migrating at ∼20 kD that comigrated with MLC (Fig. 3 A). We then subjected these same lysates to immunoprecipitation using an antibody directed to myosin IIB since this form of myosin is present in COS cells (Klemke, R., unpublished data). This antibody precipitated the 20-kD light chains from these cells, which showed increased phosphate incorporation in the MEK+ cells relative to vector transfected control cells Fig. 3 B. In addition, the phosphorylation of this protein was blocked by pretreatment of MEK+ cells with the MEK inhibitor PD98059 (Fig. 3).

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