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

Related in: MedlinePlus

MLCK activity is required for MAP kinase-induced phosphorylation of MLC and cell migration. (A, upper panel) COS cells  were transfected with empty vector or mutationally active MEK+ along with an HA-tagged ERK2 reporter construct. Cells were allowed to migrate on a collagen substrate for 3 h in the presence or absence of the MLCK inhibitor KT5926 (10 μM) as described in  Materials and Methods. Cell migration was enumerated as described above. (Lower panel) Lysates prepared from these COS cells metabolically labeled with 32P were examined by SDS-PAGE and autoradiography. The position of HA-tagged ERK and MLC are denoted  by arrows. (B, upper panel) COS cells were transfected with mutationally active MEK+ and/or MLCK(Mut) and allowed to migrate on a  collagen substrate as described in Materials and Methods. Cell migration was quantified by counting the number of migrant cells per  high powered microscopic field as described above. (Lower panel) Lysates prepared from 32P metabolically labeled COS cells transfected as above were examined for phosphorylation of HA-ERK and MLC. 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 7: MLCK activity is required for MAP kinase-induced phosphorylation of MLC and cell migration. (A, upper panel) COS cells were transfected with empty vector or mutationally active MEK+ along with an HA-tagged ERK2 reporter construct. Cells were allowed to migrate on a collagen substrate for 3 h in the presence or absence of the MLCK inhibitor KT5926 (10 μM) as described in Materials and Methods. Cell migration was enumerated as described above. (Lower panel) Lysates prepared from these COS cells metabolically labeled with 32P were examined by SDS-PAGE and autoradiography. The position of HA-tagged ERK and MLC are denoted by arrows. (B, upper panel) COS cells were transfected with mutationally active MEK+ and/or MLCK(Mut) and allowed to migrate on a collagen substrate as described in Materials and Methods. Cell migration was quantified by counting the number of migrant cells per high powered microscopic field as described above. (Lower panel) Lysates prepared from 32P metabolically labeled COS cells transfected as above were examined for phosphorylation of HA-ERK and MLC. The result shown is a representative experiment from at least three independent experiments.

Mentions: To establish a role for MLCK in the MAP kinase–induced cell migration response, COS cells transfected with MEK+ were allowed to migrate in the presence or absence of the MLCK inhibitor KT5926 (Nakanishi et al., 1990). As shown in Fig. 7 A, upper panel, KT5926 not only blocked the MEKinduced migration response but also prevented the phosphorylation of MLC in these cells in response to the expression of MEK+ (Fig. 7 A, lower panel). These findings suggest that MLCK plays a pivotal role in the MAP kinase–induced cell migration response.


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)

MLCK activity is required for MAP kinase-induced phosphorylation of MLC and cell migration. (A, upper panel) COS cells  were transfected with empty vector or mutationally active MEK+ along with an HA-tagged ERK2 reporter construct. Cells were allowed to migrate on a collagen substrate for 3 h in the presence or absence of the MLCK inhibitor KT5926 (10 μM) as described in  Materials and Methods. Cell migration was enumerated as described above. (Lower panel) Lysates prepared from these COS cells metabolically labeled with 32P were examined by SDS-PAGE and autoradiography. The position of HA-tagged ERK and MLC are denoted  by arrows. (B, upper panel) COS cells were transfected with mutationally active MEK+ and/or MLCK(Mut) and allowed to migrate on a  collagen substrate as described in Materials and Methods. Cell migration was quantified by counting the number of migrant cells per  high powered microscopic field as described above. (Lower panel) Lysates prepared from 32P metabolically labeled COS cells transfected as above were examined for phosphorylation of HA-ERK and MLC. 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 7: MLCK activity is required for MAP kinase-induced phosphorylation of MLC and cell migration. (A, upper panel) COS cells were transfected with empty vector or mutationally active MEK+ along with an HA-tagged ERK2 reporter construct. Cells were allowed to migrate on a collagen substrate for 3 h in the presence or absence of the MLCK inhibitor KT5926 (10 μM) as described in Materials and Methods. Cell migration was enumerated as described above. (Lower panel) Lysates prepared from these COS cells metabolically labeled with 32P were examined by SDS-PAGE and autoradiography. The position of HA-tagged ERK and MLC are denoted by arrows. (B, upper panel) COS cells were transfected with mutationally active MEK+ and/or MLCK(Mut) and allowed to migrate on a collagen substrate as described in Materials and Methods. Cell migration was quantified by counting the number of migrant cells per high powered microscopic field as described above. (Lower panel) Lysates prepared from 32P metabolically labeled COS cells transfected as above were examined for phosphorylation of HA-ERK and MLC. The result shown is a representative experiment from at least three independent experiments.
Mentions: To establish a role for MLCK in the MAP kinase–induced cell migration response, COS cells transfected with MEK+ were allowed to migrate in the presence or absence of the MLCK inhibitor KT5926 (Nakanishi et al., 1990). As shown in Fig. 7 A, upper panel, KT5926 not only blocked the MEKinduced migration response but also prevented the phosphorylation of MLC in these cells in response to the expression of MEK+ (Fig. 7 A, lower panel). These findings suggest that MLCK plays a pivotal role in the MAP kinase–induced cell migration response.

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