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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 directly phosphorylates MLCK, which  leads to increased MLC phosphorylation in vitro. (A) Purified  MLCK was monitored for phosphorylation in the presence or absence of purified activated MAP kinase for various times in an in  vitro kinase assay as described in Materials and Methods. Note  that there is no detectable phosphorylation of MLCK in the absence of MAP kinase. The result shown is a representative experiment from at least three independent experiments. (B) Purified  MLCK phosphorylated by MAP kinase for 40 min was tested for  its ability to phosphorylate MLC in an in vitro kinase assay in the  presence or absence of Ca/calmodulin. Note that MLC was not  phosphorylated by purified activated MAP kinase in these experiments. The result shown is a representative experiment from at  least three independent experiments. (C) MAP kinase was allowed to phosphorylate MLCK as described in vitro for 10 or 40  min as described in Materials and Methods. The MLCK treated  in the presence or absence of ERK was then allowed to phosphorylate MLC for 4 min in the presence of calcium and calmodulin as described in Materials and Methods. Data are expressed as percent of  control MLCK activity (i.e., MLCK incubated for 10 min in the absence of ERK). Each point represents the mean ± SE of at least three  experiments. The results are significant P < 0.005.
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Figure 5: MAP kinase directly phosphorylates MLCK, which leads to increased MLC phosphorylation in vitro. (A) Purified MLCK was monitored for phosphorylation in the presence or absence of purified activated MAP kinase for various times in an in vitro kinase assay as described in Materials and Methods. Note that there is no detectable phosphorylation of MLCK in the absence of MAP kinase. The result shown is a representative experiment from at least three independent experiments. (B) Purified MLCK phosphorylated by MAP kinase for 40 min was tested for its ability to phosphorylate MLC in an in vitro kinase assay in the presence or absence of Ca/calmodulin. Note that MLC was not phosphorylated by purified activated MAP kinase in these experiments. The result shown is a representative experiment from at least three independent experiments. (C) MAP kinase was allowed to phosphorylate MLCK as described in vitro for 10 or 40 min as described in Materials and Methods. The MLCK treated in the presence or absence of ERK was then allowed to phosphorylate MLC for 4 min in the presence of calcium and calmodulin as described in Materials and Methods. Data are expressed as percent of control MLCK activity (i.e., MLCK incubated for 10 min in the absence of ERK). Each point represents the mean ± SE of at least three experiments. The results are significant P < 0.005.

Mentions: To establish whether MLCK is a direct substrate for MAP kinase, we examined the ability of constitutively active MAP kinase (ERK1 or ERK2) to phosphorylate purified MLCK in an in vitro kinase assay. As shown in Fig. 5 A, purified active MAP kinase promoted a time-dependent increase in MLCK phosphorylation. This event could not be attributed to MLCK autophosphorylation since, in the absence of MAP kinase, we observed no significant phosphorylation of MLCK under the same reaction conditions (Fig. 5 A). MLCK phosphorylated with ERK2 for 40 min was then allowed to incubate with MLC in the presence or absence of calcium/calmodulin (Fig. 5 B). In this case, MLC phosphorylation was shown to depend on the presence of calcium/calmodulin. In addition, ERK2 in the absence of MLCK was incapable of phosphorylating MLC (Fig. 5 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 directly phosphorylates MLCK, which  leads to increased MLC phosphorylation in vitro. (A) Purified  MLCK was monitored for phosphorylation in the presence or absence of purified activated MAP kinase for various times in an in  vitro kinase assay as described in Materials and Methods. Note  that there is no detectable phosphorylation of MLCK in the absence of MAP kinase. The result shown is a representative experiment from at least three independent experiments. (B) Purified  MLCK phosphorylated by MAP kinase for 40 min was tested for  its ability to phosphorylate MLC in an in vitro kinase assay in the  presence or absence of Ca/calmodulin. Note that MLC was not  phosphorylated by purified activated MAP kinase in these experiments. The result shown is a representative experiment from at  least three independent experiments. (C) MAP kinase was allowed to phosphorylate MLCK as described in vitro for 10 or 40  min as described in Materials and Methods. The MLCK treated  in the presence or absence of ERK was then allowed to phosphorylate MLC for 4 min in the presence of calcium and calmodulin as described in Materials and Methods. Data are expressed as percent of  control MLCK activity (i.e., MLCK incubated for 10 min in the absence of ERK). Each point represents the mean ± SE of at least three  experiments. The results are significant P < 0.005.
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Related In: Results  -  Collection

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

Figure 5: MAP kinase directly phosphorylates MLCK, which leads to increased MLC phosphorylation in vitro. (A) Purified MLCK was monitored for phosphorylation in the presence or absence of purified activated MAP kinase for various times in an in vitro kinase assay as described in Materials and Methods. Note that there is no detectable phosphorylation of MLCK in the absence of MAP kinase. The result shown is a representative experiment from at least three independent experiments. (B) Purified MLCK phosphorylated by MAP kinase for 40 min was tested for its ability to phosphorylate MLC in an in vitro kinase assay in the presence or absence of Ca/calmodulin. Note that MLC was not phosphorylated by purified activated MAP kinase in these experiments. The result shown is a representative experiment from at least three independent experiments. (C) MAP kinase was allowed to phosphorylate MLCK as described in vitro for 10 or 40 min as described in Materials and Methods. The MLCK treated in the presence or absence of ERK was then allowed to phosphorylate MLC for 4 min in the presence of calcium and calmodulin as described in Materials and Methods. Data are expressed as percent of control MLCK activity (i.e., MLCK incubated for 10 min in the absence of ERK). Each point represents the mean ± SE of at least three experiments. The results are significant P < 0.005.
Mentions: To establish whether MLCK is a direct substrate for MAP kinase, we examined the ability of constitutively active MAP kinase (ERK1 or ERK2) to phosphorylate purified MLCK in an in vitro kinase assay. As shown in Fig. 5 A, purified active MAP kinase promoted a time-dependent increase in MLCK phosphorylation. This event could not be attributed to MLCK autophosphorylation since, in the absence of MAP kinase, we observed no significant phosphorylation of MLCK under the same reaction conditions (Fig. 5 A). MLCK phosphorylated with ERK2 for 40 min was then allowed to incubate with MLC in the presence or absence of calcium/calmodulin (Fig. 5 B). In this case, MLC phosphorylation was shown to depend on the presence of calcium/calmodulin. In addition, ERK2 in the absence of MLCK was incapable of phosphorylating MLC (Fig. 5 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
Related in: MedlinePlus