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PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation.

Slack-Davis JK, Eblen ST, Zecevic M, Boerner SA, Tarcsafalvi A, Diaz HB, Marshall MS, Weber MJ, Parsons JT, Catling AD - J. Cell Biol. (2003)

Bottom Line: Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM.Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling.We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908, USA.

ABSTRACT
Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

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Src inhibition alters the location of phospho-MAPK. REF52 cells were suspended in the presence or absence of 50 μM PP2 and plated on FN for 1 h in the continued presence or absence of PP2 before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate peripheral adhesion complexes containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.
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fig9: Src inhibition alters the location of phospho-MAPK. REF52 cells were suspended in the presence or absence of 50 μM PP2 and plated on FN for 1 h in the continued presence or absence of PP2 before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate peripheral adhesion complexes containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.

Mentions: Because Src-dependent MEK1 S298 phosphorylation is required for adhesion-mediated activation of MEK (Fig. 5) and subsequent MAPK activation (Eblen et al., 2002), we examined the effect of Src inhibition on phospho-MAPK localization in REF52 cells. Cells were suspended for 1 h and allowed to spread on FN for 1 h in the continued presence or absence of PP2 and immunostained for phospho-MAPK (red) and paxillin (green). Consistent with a previous report (Fincham et al., 2000), inhibition of Src activity had no effect on the localization of phospho-MAPK to focal adhesions (Fig. 9, arrowheads). However, PP2 decreased the extent of phospho-MAPK staining in peripheral structures reminiscent of Rac-induced adhesion complexes. Indeed, only 22% of the cells treated with PP2 exhibited peripheral adhesion complexes containing phospho-MAPK, whereas almost all (98%) of the untreated REF52 cells displayed peripheral adhesion complexes rich in phospho-MAPK staining (Fig. 9, arrows). These observations indicate that, in addition to decreasing MAPK activation, inhibition of Src activity reduces the pool of activated MAPK recruited to newly formed adhesion complexes.


PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation.

Slack-Davis JK, Eblen ST, Zecevic M, Boerner SA, Tarcsafalvi A, Diaz HB, Marshall MS, Weber MJ, Parsons JT, Catling AD - J. Cell Biol. (2003)

Src inhibition alters the location of phospho-MAPK. REF52 cells were suspended in the presence or absence of 50 μM PP2 and plated on FN for 1 h in the continued presence or absence of PP2 before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate peripheral adhesion complexes containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172784&req=5

fig9: Src inhibition alters the location of phospho-MAPK. REF52 cells were suspended in the presence or absence of 50 μM PP2 and plated on FN for 1 h in the continued presence or absence of PP2 before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate peripheral adhesion complexes containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.
Mentions: Because Src-dependent MEK1 S298 phosphorylation is required for adhesion-mediated activation of MEK (Fig. 5) and subsequent MAPK activation (Eblen et al., 2002), we examined the effect of Src inhibition on phospho-MAPK localization in REF52 cells. Cells were suspended for 1 h and allowed to spread on FN for 1 h in the continued presence or absence of PP2 and immunostained for phospho-MAPK (red) and paxillin (green). Consistent with a previous report (Fincham et al., 2000), inhibition of Src activity had no effect on the localization of phospho-MAPK to focal adhesions (Fig. 9, arrowheads). However, PP2 decreased the extent of phospho-MAPK staining in peripheral structures reminiscent of Rac-induced adhesion complexes. Indeed, only 22% of the cells treated with PP2 exhibited peripheral adhesion complexes containing phospho-MAPK, whereas almost all (98%) of the untreated REF52 cells displayed peripheral adhesion complexes rich in phospho-MAPK staining (Fig. 9, arrows). These observations indicate that, in addition to decreasing MAPK activation, inhibition of Src activity reduces the pool of activated MAPK recruited to newly formed adhesion complexes.

Bottom Line: Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM.Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling.We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908, USA.

ABSTRACT
Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Show MeSH
Related in: MedlinePlus