Limits...
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.

Show MeSH
MAP kinase activity is associated with increased phosphorylation of  MLCK. (A) MLCK was immunoprecipitated from an aliquot of COS-7 cell lysates described in Fig. 3 and resolved  by SDS-PAGE and autoradiography or (B) Western blotted with an MLCK-specific  antibody. The phosphorylated  proteins of ∼160 and 200 kD  represent MLCK isoforms present in COS-7 cells, whereas the 97-kD protein may represent a break down product of MLCK. The result  shown is a representative experiment from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139771&req=5

Figure 4: MAP kinase activity is associated with increased phosphorylation of MLCK. (A) MLCK was immunoprecipitated from an aliquot of COS-7 cell lysates described in Fig. 3 and resolved by SDS-PAGE and autoradiography or (B) Western blotted with an MLCK-specific antibody. The phosphorylated proteins of ∼160 and 200 kD represent MLCK isoforms present in COS-7 cells, whereas the 97-kD protein may represent a break down product of MLCK. The result shown is a representative experiment from at least three independent experiments.

Mentions: Phosphorylation of MLC by the Ca2+/calmodulin-dependent enzyme, MLCK, is critical for cell migration. This reaction promotes increased myosin filament formation leading to its association with actin filaments and stimulates the actin-activated ATPase activity of myosin II. In addition, the fact that MLCK contains multiple MAP kinase consensus phosphorylation sites (P-x-S[T]-P) (Shoemaker et al., 1990) prompted us to examine the possibility that MLCK could be directly phosphorylated by MAP kinase. Therefore, MLCK was immunoprecipitated from MEK+ or control transfectants that had been metabolically labeled with [32P]orthophosphate. As shown in Fig. 4 A, anti-MLCK antibodies precipitated radiolabeled proteins of 160–200 kD, which represents isoforms of MLCK found in COS-7 cells and an additional (∼97 kD) breakdown product of MLCK as previously described (Gallagher et al., 1995). In contrast, cells transfected with a control vector express these proteins with minimal phosphate incorporation (Fig. 4 A). The phosphorylation of MLCK appeared to be caused by the activation of MAP kinase in these cells since MEK+ cells treated with the MEK inhibitor showed a loss of the MLCK phosphorylation. The enhanced phosphorylation of MLCK in MEK+ cells could not be explained by increased MLCK protein expression since immunoblotting experiments revealed that lysates from these and control cells contained the same level of MLCK (Fig. 4 B).


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 phosphorylation of  MLCK. (A) MLCK was immunoprecipitated from an aliquot of COS-7 cell lysates described in Fig. 3 and resolved  by SDS-PAGE and autoradiography or (B) Western blotted with an MLCK-specific  antibody. The phosphorylated  proteins of ∼160 and 200 kD  represent MLCK isoforms present in COS-7 cells, whereas the 97-kD protein may represent a break down product of MLCK. 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 4: MAP kinase activity is associated with increased phosphorylation of MLCK. (A) MLCK was immunoprecipitated from an aliquot of COS-7 cell lysates described in Fig. 3 and resolved by SDS-PAGE and autoradiography or (B) Western blotted with an MLCK-specific antibody. The phosphorylated proteins of ∼160 and 200 kD represent MLCK isoforms present in COS-7 cells, whereas the 97-kD protein may represent a break down product of MLCK. The result shown is a representative experiment from at least three independent experiments.
Mentions: Phosphorylation of MLC by the Ca2+/calmodulin-dependent enzyme, MLCK, is critical for cell migration. This reaction promotes increased myosin filament formation leading to its association with actin filaments and stimulates the actin-activated ATPase activity of myosin II. In addition, the fact that MLCK contains multiple MAP kinase consensus phosphorylation sites (P-x-S[T]-P) (Shoemaker et al., 1990) prompted us to examine the possibility that MLCK could be directly phosphorylated by MAP kinase. Therefore, MLCK was immunoprecipitated from MEK+ or control transfectants that had been metabolically labeled with [32P]orthophosphate. As shown in Fig. 4 A, anti-MLCK antibodies precipitated radiolabeled proteins of 160–200 kD, which represents isoforms of MLCK found in COS-7 cells and an additional (∼97 kD) breakdown product of MLCK as previously described (Gallagher et al., 1995). In contrast, cells transfected with a control vector express these proteins with minimal phosphate incorporation (Fig. 4 A). The phosphorylation of MLCK appeared to be caused by the activation of MAP kinase in these cells since MEK+ cells treated with the MEK inhibitor showed a loss of the MLCK phosphorylation. The enhanced phosphorylation of MLCK in MEK+ cells could not be explained by increased MLCK protein expression since immunoblotting experiments revealed that lysates from these and control cells contained the same level of MLCK (Fig. 4 B).

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