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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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FAK signaling regulates MEK1 S298 phosphorylation. (A) FAK- fibroblasts or cells from wild-type littermate controls were suspended (S), plated on FN for 5, 15, or 45 min, or remained adherent (A). Whole cell lysates were blotted with p-S298MEK1, MEK1, p-MAPK, or ERK2 antisera. (B) FAK- fibroblasts transfected with myc-tagged wild-type FAK, FAK Y397F, or empty vector control together with HA-tagged wild-type MEK1 were suspended (S) and replated on FN for 5, 10, or 20 min. Immunoprecipitates were formed using HA antiserum and blotted for p-S298MEK1 or MEK1. FAK expression was determined from whole cell lysates of transfected cells using Myc antiserum. FAK autophosphorylation was determined by immunoblotting with Y397 FAK phospho-specific antibodies.
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fig6: FAK signaling regulates MEK1 S298 phosphorylation. (A) FAK- fibroblasts or cells from wild-type littermate controls were suspended (S), plated on FN for 5, 15, or 45 min, or remained adherent (A). Whole cell lysates were blotted with p-S298MEK1, MEK1, p-MAPK, or ERK2 antisera. (B) FAK- fibroblasts transfected with myc-tagged wild-type FAK, FAK Y397F, or empty vector control together with HA-tagged wild-type MEK1 were suspended (S) and replated on FN for 5, 10, or 20 min. Immunoprecipitates were formed using HA antiserum and blotted for p-S298MEK1 or MEK1. FAK expression was determined from whole cell lysates of transfected cells using Myc antiserum. FAK autophosphorylation was determined by immunoblotting with Y397 FAK phospho-specific antibodies.

Mentions: FAK and Src regulate a well-defined pathway that becomes activated after integrin engagement (Parsons et al., 2000). In addition, FAK/Src signaling has been shown to play a role in FN-stimulated MAPK activation (Schlaepfer and Hunter, 1996; Schlaepfer et al., 1994). We examined the role of this pathway in regulating MEK1 S298 phosphorylation using FAK-deficient cells or PP2, an inhibitor of Src family kinases. FAK- fibroblasts or controls from wild-type littermates were placed in suspension for 1 h and stimulated to spread on FN for the indicated times (Fig. 6 A). Western blot analysis of whole cell lysates using anti–p-S298 MEK1 demonstrated decreased levels (∼70% wild-type levels determined by densitometry) and delayed time course of MEK1 S298 phosphorylation in FAK- cells compared with FAK-expressing wild-type fibroblasts (Fig. 6 A). Similarly, decreasing FAK protein levels in REF52 cells using siRNAs also resulted in decreased and delayed MEK1 S298 phosphorylation upon adhesion of these cells to FN (unpublished observations; Fig. S1). In both FAK- and -expressing cells, MAPK phosphorylation paralleled MEK1 S298 phosphorylation (Fig. 6 A).


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)

FAK signaling regulates MEK1 S298 phosphorylation. (A) FAK- fibroblasts or cells from wild-type littermate controls were suspended (S), plated on FN for 5, 15, or 45 min, or remained adherent (A). Whole cell lysates were blotted with p-S298MEK1, MEK1, p-MAPK, or ERK2 antisera. (B) FAK- fibroblasts transfected with myc-tagged wild-type FAK, FAK Y397F, or empty vector control together with HA-tagged wild-type MEK1 were suspended (S) and replated on FN for 5, 10, or 20 min. Immunoprecipitates were formed using HA antiserum and blotted for p-S298MEK1 or MEK1. FAK expression was determined from whole cell lysates of transfected cells using Myc antiserum. FAK autophosphorylation was determined by immunoblotting with Y397 FAK phospho-specific antibodies.
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Related In: Results  -  Collection

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fig6: FAK signaling regulates MEK1 S298 phosphorylation. (A) FAK- fibroblasts or cells from wild-type littermate controls were suspended (S), plated on FN for 5, 15, or 45 min, or remained adherent (A). Whole cell lysates were blotted with p-S298MEK1, MEK1, p-MAPK, or ERK2 antisera. (B) FAK- fibroblasts transfected with myc-tagged wild-type FAK, FAK Y397F, or empty vector control together with HA-tagged wild-type MEK1 were suspended (S) and replated on FN for 5, 10, or 20 min. Immunoprecipitates were formed using HA antiserum and blotted for p-S298MEK1 or MEK1. FAK expression was determined from whole cell lysates of transfected cells using Myc antiserum. FAK autophosphorylation was determined by immunoblotting with Y397 FAK phospho-specific antibodies.
Mentions: FAK and Src regulate a well-defined pathway that becomes activated after integrin engagement (Parsons et al., 2000). In addition, FAK/Src signaling has been shown to play a role in FN-stimulated MAPK activation (Schlaepfer and Hunter, 1996; Schlaepfer et al., 1994). We examined the role of this pathway in regulating MEK1 S298 phosphorylation using FAK-deficient cells or PP2, an inhibitor of Src family kinases. FAK- fibroblasts or controls from wild-type littermates were placed in suspension for 1 h and stimulated to spread on FN for the indicated times (Fig. 6 A). Western blot analysis of whole cell lysates using anti–p-S298 MEK1 demonstrated decreased levels (∼70% wild-type levels determined by densitometry) and delayed time course of MEK1 S298 phosphorylation in FAK- cells compared with FAK-expressing wild-type fibroblasts (Fig. 6 A). Similarly, decreasing FAK protein levels in REF52 cells using siRNAs also resulted in decreased and delayed MEK1 S298 phosphorylation upon adhesion of these cells to FN (unpublished observations; Fig. S1). In both FAK- and -expressing cells, MAPK phosphorylation paralleled MEK1 S298 phosphorylation (Fig. 6 A).

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