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

MAP kinase (ERK1  and ERK2) is required for cell  migration but not adhesion  or spreading on extracellular  matrix proteins. (A, lower  panel) Cells were allowed to  adhere for 1 h or migrate for  6 h on a collagen substrate  after exposure to MAP kinase–specific antisense or a  scrambled control oligonucleotide. Migration studies  were performed using Transwell migration chambers  coated with collagen as described in Materials and Methods. (Upper panel) Total cell  lysates (50 μg) prepared from  an aliquot of those FG cells  analyzed for migration/adhesion were immunoblotted with  antibodies to Raf-1, actin, or  ERK proteins. The upper  band is a nonspecific protein  recognized by the rabbit polyclonal ERK antisera. The result shown is a representative  experiment from at least three  independent experiments. (B,  upper panel) Phosphotyrosine immunoblot or kinase  activity of ERK1 and ERK2  proteins immunoprecipitated  from cells either pretreated in  the presence or absence of  the MEK inhibitor PD98059  (25 μM) and allowed to attach to poly-l-lysine or collagen-coated dishes for 30  min. The ERK immunoprecipitates were also examined  for their ability to phosphorylate MBP in an in vitro kinase  assay as described in Materials and Methods. (Lower  panel) Cells treated in the  presence or absence of the  MEK inhibitor as above were  allowed to attach for 1 h or  migrate for 6 h on a collagen  substrate and quantified as  described in Materials and  Methods. The result shown is  a representative experiment  from at least three independent experiments. (C) Nomarski photomicrograph (600×)  of FG cells pretreated for 6 h  in the presence or absence  (control) of the MEK inhibitor (100 μM) and allowed to  attach and spread on collagen-coated coverslips for  10 or 60 min. The result  shown is a representative experiment from at least three  independent experiments.  Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139771&req=5

Figure 1: MAP kinase (ERK1 and ERK2) is required for cell migration but not adhesion or spreading on extracellular matrix proteins. (A, lower panel) Cells were allowed to adhere for 1 h or migrate for 6 h on a collagen substrate after exposure to MAP kinase–specific antisense or a scrambled control oligonucleotide. Migration studies were performed using Transwell migration chambers coated with collagen as described in Materials and Methods. (Upper panel) Total cell lysates (50 μg) prepared from an aliquot of those FG cells analyzed for migration/adhesion were immunoblotted with antibodies to Raf-1, actin, or ERK proteins. The upper band is a nonspecific protein recognized by the rabbit polyclonal ERK antisera. The result shown is a representative experiment from at least three independent experiments. (B, upper panel) Phosphotyrosine immunoblot or kinase activity of ERK1 and ERK2 proteins immunoprecipitated from cells either pretreated in the presence or absence of the MEK inhibitor PD98059 (25 μM) and allowed to attach to poly-l-lysine or collagen-coated dishes for 30 min. The ERK immunoprecipitates were also examined for their ability to phosphorylate MBP in an in vitro kinase assay as described in Materials and Methods. (Lower panel) Cells treated in the presence or absence of the MEK inhibitor as above were allowed to attach for 1 h or migrate for 6 h on a collagen substrate and quantified as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments. (C) Nomarski photomicrograph (600×) of FG cells pretreated for 6 h in the presence or absence (control) of the MEK inhibitor (100 μM) and allowed to attach and spread on collagen-coated coverslips for 10 or 60 min. The result shown is a representative experiment from at least three independent experiments. Bar, 10 μm.

Mentions: To examine a potential role of MAP kinase in cell migration, we examined FG carcinoma cells, which readily migrate on a collagen substrate using integrin α2β1 (Leavesley et al., 1992; Klemke et al., 1994). FG cells were pretreated with antisense oligonucleotides specific for ERK1 and ERK2 (Sale et al., 1995) or a scrambled control oligonucleotide and allowed to attach, spread, and migrate on a collagen substrate. As shown in Fig. 1 A, upper panel, exposure of cells to antisense but not a control oligonucleotide reduced ERK1 and ERK2 protein levels by ∼90% yet had no effect on expression of other proteins in these cells, as determined by Western blotting with antibodies to Raf-1 and actin or a nonspecific protein recognized by the rabbit polyclonal antibodies to ERK1 and ERK2. Cells treated with the antisense oligonucleotide readily attached and spread (data not shown) on collagen, but showed 75% decreased migration (Fig. 1 A, lower panel) relative to cells treated with the control oligonucleotide. These findings suggest that MAP kinase influences cell migration yet has little apparent effect on adhesion or spreading on a collagen substrate.


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 (ERK1  and ERK2) is required for cell  migration but not adhesion  or spreading on extracellular  matrix proteins. (A, lower  panel) Cells were allowed to  adhere for 1 h or migrate for  6 h on a collagen substrate  after exposure to MAP kinase–specific antisense or a  scrambled control oligonucleotide. Migration studies  were performed using Transwell migration chambers  coated with collagen as described in Materials and Methods. (Upper panel) Total cell  lysates (50 μg) prepared from  an aliquot of those FG cells  analyzed for migration/adhesion were immunoblotted with  antibodies to Raf-1, actin, or  ERK proteins. The upper  band is a nonspecific protein  recognized by the rabbit polyclonal ERK antisera. The result shown is a representative  experiment from at least three  independent experiments. (B,  upper panel) Phosphotyrosine immunoblot or kinase  activity of ERK1 and ERK2  proteins immunoprecipitated  from cells either pretreated in  the presence or absence of  the MEK inhibitor PD98059  (25 μM) and allowed to attach to poly-l-lysine or collagen-coated dishes for 30  min. The ERK immunoprecipitates were also examined  for their ability to phosphorylate MBP in an in vitro kinase  assay as described in Materials and Methods. (Lower  panel) Cells treated in the  presence or absence of the  MEK inhibitor as above were  allowed to attach for 1 h or  migrate for 6 h on a collagen  substrate and quantified as  described in Materials and  Methods. The result shown is  a representative experiment  from at least three independent experiments. (C) Nomarski photomicrograph (600×)  of FG cells pretreated for 6 h  in the presence or absence  (control) of the MEK inhibitor (100 μM) and allowed to  attach and spread on collagen-coated coverslips for  10 or 60 min. The result  shown is a representative experiment from at least three  independent experiments.  Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: MAP kinase (ERK1 and ERK2) is required for cell migration but not adhesion or spreading on extracellular matrix proteins. (A, lower panel) Cells were allowed to adhere for 1 h or migrate for 6 h on a collagen substrate after exposure to MAP kinase–specific antisense or a scrambled control oligonucleotide. Migration studies were performed using Transwell migration chambers coated with collagen as described in Materials and Methods. (Upper panel) Total cell lysates (50 μg) prepared from an aliquot of those FG cells analyzed for migration/adhesion were immunoblotted with antibodies to Raf-1, actin, or ERK proteins. The upper band is a nonspecific protein recognized by the rabbit polyclonal ERK antisera. The result shown is a representative experiment from at least three independent experiments. (B, upper panel) Phosphotyrosine immunoblot or kinase activity of ERK1 and ERK2 proteins immunoprecipitated from cells either pretreated in the presence or absence of the MEK inhibitor PD98059 (25 μM) and allowed to attach to poly-l-lysine or collagen-coated dishes for 30 min. The ERK immunoprecipitates were also examined for their ability to phosphorylate MBP in an in vitro kinase assay as described in Materials and Methods. (Lower panel) Cells treated in the presence or absence of the MEK inhibitor as above were allowed to attach for 1 h or migrate for 6 h on a collagen substrate and quantified as described in Materials and Methods. The result shown is a representative experiment from at least three independent experiments. (C) Nomarski photomicrograph (600×) of FG cells pretreated for 6 h in the presence or absence (control) of the MEK inhibitor (100 μM) and allowed to attach and spread on collagen-coated coverslips for 10 or 60 min. The result shown is a representative experiment from at least three independent experiments. Bar, 10 μm.
Mentions: To examine a potential role of MAP kinase in cell migration, we examined FG carcinoma cells, which readily migrate on a collagen substrate using integrin α2β1 (Leavesley et al., 1992; Klemke et al., 1994). FG cells were pretreated with antisense oligonucleotides specific for ERK1 and ERK2 (Sale et al., 1995) or a scrambled control oligonucleotide and allowed to attach, spread, and migrate on a collagen substrate. As shown in Fig. 1 A, upper panel, exposure of cells to antisense but not a control oligonucleotide reduced ERK1 and ERK2 protein levels by ∼90% yet had no effect on expression of other proteins in these cells, as determined by Western blotting with antibodies to Raf-1 and actin or a nonspecific protein recognized by the rabbit polyclonal antibodies to ERK1 and ERK2. Cells treated with the antisense oligonucleotide readily attached and spread (data not shown) on collagen, but showed 75% decreased migration (Fig. 1 A, lower panel) relative to cells treated with the control oligonucleotide. These findings suggest that MAP kinase influences cell migration yet has little apparent effect on adhesion or spreading on a collagen substrate.

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