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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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Adhesion stimulates MAPK and MEK phosphorylation. REF52 cells were either continuously adherent (A) or suspended (S) and plated on FN for 5, 10, 20, or 40 min. Whole cell lysates were blotted with antiserum specific for (A) phosphorylated MAPK (p-MAPK; top) or ERK2 (bottom), or (B) MEK1 phosphorylated on S218/S222 (p-S218/222MEK1; top) or MEK1 (bottom). (C) REF52 cells were suspended for 1 h and plated on FN for 1 h before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate focal complex-like structures containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.
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fig1: Adhesion stimulates MAPK and MEK phosphorylation. REF52 cells were either continuously adherent (A) or suspended (S) and plated on FN for 5, 10, 20, or 40 min. Whole cell lysates were blotted with antiserum specific for (A) phosphorylated MAPK (p-MAPK; top) or ERK2 (bottom), or (B) MEK1 phosphorylated on S218/S222 (p-S218/222MEK1; top) or MEK1 (bottom). (C) REF52 cells were suspended for 1 h and plated on FN for 1 h before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate focal complex-like structures containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.

Mentions: Cell adhesion to FN in the absence of growth factors activates both PAK1 and MAPK (Figs. 1 A and 8 B; Aplin et al., 1999; Bagrodia and Cerione, 1999). Suspension of REF52 cells for 1 h in serum-free media greatly reduced phosphorylation of MAPK on the activating sites (i.e., T183/Y185 of ERK2) (Fig. 1, A and B). Replating suspended cells on FN stimulated both MEK activation site phosphorylation (S218/S222) and MAPK phosphorylation within 10 min, with maximal MEK phosphorylation on the activating sites occurring between 5 and 10 min (Fig. 1, A and B). To determine the localization of this active pool of MAPK, REF52 cells suspended for 1 h in serum-free media were replated on FN for 1 h in the absence of serum. Immunostaining with an antibody to the phosphorylated form of MAPK revealed phospho-MAPK colocalized with paxillin in well-defined adhesions (Fig. 1 C, arrowhead). In addition, prominent MAPK staining in peripheral structures resembling Rac-induced focal complexes was observed. These structures were distinguished from focal adhesions in that they did not contain significant levels of paxillin staining (Fig. 1 C, arrow). Cells spreading for shorter periods of time showed poorly organized phospho-MAPK staining (unpublished data).


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)

Adhesion stimulates MAPK and MEK phosphorylation. REF52 cells were either continuously adherent (A) or suspended (S) and plated on FN for 5, 10, 20, or 40 min. Whole cell lysates were blotted with antiserum specific for (A) phosphorylated MAPK (p-MAPK; top) or ERK2 (bottom), or (B) MEK1 phosphorylated on S218/S222 (p-S218/222MEK1; top) or MEK1 (bottom). (C) REF52 cells were suspended for 1 h and plated on FN for 1 h before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate focal complex-like structures containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.
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Related In: Results  -  Collection

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

fig1: Adhesion stimulates MAPK and MEK phosphorylation. REF52 cells were either continuously adherent (A) or suspended (S) and plated on FN for 5, 10, 20, or 40 min. Whole cell lysates were blotted with antiserum specific for (A) phosphorylated MAPK (p-MAPK; top) or ERK2 (bottom), or (B) MEK1 phosphorylated on S218/S222 (p-S218/222MEK1; top) or MEK1 (bottom). (C) REF52 cells were suspended for 1 h and plated on FN for 1 h before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate focal complex-like structures containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.
Mentions: Cell adhesion to FN in the absence of growth factors activates both PAK1 and MAPK (Figs. 1 A and 8 B; Aplin et al., 1999; Bagrodia and Cerione, 1999). Suspension of REF52 cells for 1 h in serum-free media greatly reduced phosphorylation of MAPK on the activating sites (i.e., T183/Y185 of ERK2) (Fig. 1, A and B). Replating suspended cells on FN stimulated both MEK activation site phosphorylation (S218/S222) and MAPK phosphorylation within 10 min, with maximal MEK phosphorylation on the activating sites occurring between 5 and 10 min (Fig. 1, A and B). To determine the localization of this active pool of MAPK, REF52 cells suspended for 1 h in serum-free media were replated on FN for 1 h in the absence of serum. Immunostaining with an antibody to the phosphorylated form of MAPK revealed phospho-MAPK colocalized with paxillin in well-defined adhesions (Fig. 1 C, arrowhead). In addition, prominent MAPK staining in peripheral structures resembling Rac-induced focal complexes was observed. These structures were distinguished from focal adhesions in that they did not contain significant levels of paxillin staining (Fig. 1 C, arrow). Cells spreading for shorter periods of time showed poorly organized phospho-MAPK staining (unpublished data).

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