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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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PAK directs MEK1 S298 phosphorylation in vitro. (A) In vitro kinase assays were performed using recombinant MEK1 and recombinant PAK3 as described in Materials and methods. (B) REF52 cells were transfected with HA-tagged wild-type MEK1 or HA-MEK1 S298A, suspended for 1 h and allowed to adhere to FN for 10 min. Anti-HA immunoprecipitates were analyzed and blotted with p-S298MEK1 (top) or MEK1 (bottom).
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fig2: PAK directs MEK1 S298 phosphorylation in vitro. (A) In vitro kinase assays were performed using recombinant MEK1 and recombinant PAK3 as described in Materials and methods. (B) REF52 cells were transfected with HA-tagged wild-type MEK1 or HA-MEK1 S298A, suspended for 1 h and allowed to adhere to FN for 10 min. Anti-HA immunoprecipitates were analyzed and blotted with p-S298MEK1 (top) or MEK1 (bottom).

Mentions: The localization of phosphorylated MAPK in structures resembling Rac-induced focal complexes prompted the examination of the role of Rac and its effector PAK in adhesion-dependent MAPK regulation. As previously reported, Rac signaling to PAK synergizes with Raf to regulate MAPK activity (Frost et al., 1997). In addition, PAK has been reported to phosphorylate MEK1 on S298 in vitro (Coles and Shaw, 2002). To examine PAK phosphorylation of MEK1, an antiphosphopeptide antiserum specific for phosphorylated S298 in MEK1 (p-S298 MEK1) was generated (see Materials and methods). Recombinant group I PAK proteins (PAK1, 2, and 3) stimulated phosphorylation of kinase-defective MEK1 on a site specifically recognized by anti–p-S298 MEK1 (Fig. 2 A, not depicted). Recognition of phosphorylated MEK1 was abolished by preincubation of anti–p-S298 MEK1 with the immunizing phosphopeptide, but not with the corresponding nonphosphopeptide (unpublished data). In addition, anti–p-S298 MEK1 failed to recognize MEK1 containing an alanine substitution at position 298 (S298A; Fig. 2 B). Immunoblotting with anti–p-S298 MEK1 revealed the loss of MEK1 S298 phosphorylation in cells suspended in serum-free media (Fig. 3 A). Replating on FN (Fig. 3 A), laminin, or vitronectin (not depicted) led to a rapid increase in MEK1 S298 phosphorylation (within 5 min), which was maintained throughout the course of the assay and equivalent to levels observed in continuously adherent cells. As shown in Fig. 3 B, anti–p-S298 MEK1 selectively identified endogenous phosphorylated MEK1, but not MEK2, immunoprecipitated from cells. Although MEK2 contains a site equivalent to S298, it does not appear to be a substrate for phosphorylation by PAK (Frost et al., 1997). Finally, adhesion-dependent MEK1 S298 phosphorylation was observed in many cell lines including WI38, mouse embryo fibroblasts, CCL39, COS-1, LNCaP, and MCF7 (unpublished data), indicating the generality of MEK1 S298 phosphorylation.


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)

PAK directs MEK1 S298 phosphorylation in vitro. (A) In vitro kinase assays were performed using recombinant MEK1 and recombinant PAK3 as described in Materials and methods. (B) REF52 cells were transfected with HA-tagged wild-type MEK1 or HA-MEK1 S298A, suspended for 1 h and allowed to adhere to FN for 10 min. Anti-HA immunoprecipitates were analyzed and blotted with p-S298MEK1 (top) or MEK1 (bottom).
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Related In: Results  -  Collection

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

fig2: PAK directs MEK1 S298 phosphorylation in vitro. (A) In vitro kinase assays were performed using recombinant MEK1 and recombinant PAK3 as described in Materials and methods. (B) REF52 cells were transfected with HA-tagged wild-type MEK1 or HA-MEK1 S298A, suspended for 1 h and allowed to adhere to FN for 10 min. Anti-HA immunoprecipitates were analyzed and blotted with p-S298MEK1 (top) or MEK1 (bottom).
Mentions: The localization of phosphorylated MAPK in structures resembling Rac-induced focal complexes prompted the examination of the role of Rac and its effector PAK in adhesion-dependent MAPK regulation. As previously reported, Rac signaling to PAK synergizes with Raf to regulate MAPK activity (Frost et al., 1997). In addition, PAK has been reported to phosphorylate MEK1 on S298 in vitro (Coles and Shaw, 2002). To examine PAK phosphorylation of MEK1, an antiphosphopeptide antiserum specific for phosphorylated S298 in MEK1 (p-S298 MEK1) was generated (see Materials and methods). Recombinant group I PAK proteins (PAK1, 2, and 3) stimulated phosphorylation of kinase-defective MEK1 on a site specifically recognized by anti–p-S298 MEK1 (Fig. 2 A, not depicted). Recognition of phosphorylated MEK1 was abolished by preincubation of anti–p-S298 MEK1 with the immunizing phosphopeptide, but not with the corresponding nonphosphopeptide (unpublished data). In addition, anti–p-S298 MEK1 failed to recognize MEK1 containing an alanine substitution at position 298 (S298A; Fig. 2 B). Immunoblotting with anti–p-S298 MEK1 revealed the loss of MEK1 S298 phosphorylation in cells suspended in serum-free media (Fig. 3 A). Replating on FN (Fig. 3 A), laminin, or vitronectin (not depicted) led to a rapid increase in MEK1 S298 phosphorylation (within 5 min), which was maintained throughout the course of the assay and equivalent to levels observed in continuously adherent cells. As shown in Fig. 3 B, anti–p-S298 MEK1 selectively identified endogenous phosphorylated MEK1, but not MEK2, immunoprecipitated from cells. Although MEK2 contains a site equivalent to S298, it does not appear to be a substrate for phosphorylation by PAK (Frost et al., 1997). Finally, adhesion-dependent MEK1 S298 phosphorylation was observed in many cell lines including WI38, mouse embryo fibroblasts, CCL39, COS-1, LNCaP, and MCF7 (unpublished data), indicating the generality of MEK1 S298 phosphorylation.

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