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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-dependent c-Raf and PAK activity are affected by inhibiting Src. (A) c-Raf activity was determined from REF52 cells incubated in suspension with 50 μM PP2 or DMSO solvent control (S) and plated on FN for 5 or 10 min. Immunoprecipitates were formed using either c-Raf antiserum, or matched control antiserum raised against the GAL4 transactivation domain, and kinase assays were performed with kinase-defective GST-MEK1 as described in Materials and methods (top). A reaction blank (lane B) confirms the absence of MEK kinase activity in the substrate and c-Raf antiserum, and a reaction lacking GST-MEK1 (lane 8) confirms that MEK1 is the phosphorylated substrate in these assays. Loading controls (bottom) demonstrate similar levels of c-Raf protein in the appropriate assays. (B) PAK1 activity was determined as described above for c-Raf activity (A) except cells were also plated for 5, 10, or 20 min and immunoprecipitates were formed using PAK1 antiserum. PAK1 activity was assessed by blotting the recombinant MEK1 substrate with the p-S298 antiserum (top). The PAK1 loading control is shown in the bottom panel. Lane B denotes a reaction blank in which PAK1 antibody was incubated with lysis buffer instead of lysate before the kinase assay.
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fig8: Adhesion-dependent c-Raf and PAK activity are affected by inhibiting Src. (A) c-Raf activity was determined from REF52 cells incubated in suspension with 50 μM PP2 or DMSO solvent control (S) and plated on FN for 5 or 10 min. Immunoprecipitates were formed using either c-Raf antiserum, or matched control antiserum raised against the GAL4 transactivation domain, and kinase assays were performed with kinase-defective GST-MEK1 as described in Materials and methods (top). A reaction blank (lane B) confirms the absence of MEK kinase activity in the substrate and c-Raf antiserum, and a reaction lacking GST-MEK1 (lane 8) confirms that MEK1 is the phosphorylated substrate in these assays. Loading controls (bottom) demonstrate similar levels of c-Raf protein in the appropriate assays. (B) PAK1 activity was determined as described above for c-Raf activity (A) except cells were also plated for 5, 10, or 20 min and immunoprecipitates were formed using PAK1 antiserum. PAK1 activity was assessed by blotting the recombinant MEK1 substrate with the p-S298 antiserum (top). The PAK1 loading control is shown in the bottom panel. Lane B denotes a reaction blank in which PAK1 antibody was incubated with lysis buffer instead of lysate before the kinase assay.

Mentions: To examine whether PAK regulates adhesion-dependent MEK1 S298 phosphorylation in vivo, COS-1 or REF52 cells expressing kinase-defective PAK1 (or vector control) together with HA-tagged wild-type MEK1 were placed in suspension and replated on FN for the indicated times and S298 phosphorylation was assessed by Western blotting. Expression of kinase-defective PAK1 significantly reduced FN-stimulated S298 phosphorylation of exogenously expressed MEK1 (Fig. 4 A, not depicted), indicating that PAK activation is necessary for adhesion-dependent MEK1 S298 phosphorylation. To determine if PAK activity is sufficient to mediate MEK1 S298 phosphorylation in vivo, MEK1 S298 phosphorylation was examined in cells overexpressing activated PAK (T423E). REF52 cells expressing activated PAK together with wild-type MEK1 exhibited elevated MEK1 S298 phosphorylation whether in suspension or plated on FN (Fig. 4 B). Lastly, endogenous PAK1 immunoprecipitated from REF52 cells was activated by adhesion to FN (see Fig. 8 B), whereas endogenous PAK2 and 4 were not (not depicted). Together, these data indicate that PAK1 activity is both necessary and sufficient to stimulate MEK1 S298 phosphorylation and that PAK1-mediated phosphorylation of MEK1 on S298 is regulated by cell adhesion.


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-dependent c-Raf and PAK activity are affected by inhibiting Src. (A) c-Raf activity was determined from REF52 cells incubated in suspension with 50 μM PP2 or DMSO solvent control (S) and plated on FN for 5 or 10 min. Immunoprecipitates were formed using either c-Raf antiserum, or matched control antiserum raised against the GAL4 transactivation domain, and kinase assays were performed with kinase-defective GST-MEK1 as described in Materials and methods (top). A reaction blank (lane B) confirms the absence of MEK kinase activity in the substrate and c-Raf antiserum, and a reaction lacking GST-MEK1 (lane 8) confirms that MEK1 is the phosphorylated substrate in these assays. Loading controls (bottom) demonstrate similar levels of c-Raf protein in the appropriate assays. (B) PAK1 activity was determined as described above for c-Raf activity (A) except cells were also plated for 5, 10, or 20 min and immunoprecipitates were formed using PAK1 antiserum. PAK1 activity was assessed by blotting the recombinant MEK1 substrate with the p-S298 antiserum (top). The PAK1 loading control is shown in the bottom panel. Lane B denotes a reaction blank in which PAK1 antibody was incubated with lysis buffer instead of lysate before the kinase assay.
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Related In: Results  -  Collection

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fig8: Adhesion-dependent c-Raf and PAK activity are affected by inhibiting Src. (A) c-Raf activity was determined from REF52 cells incubated in suspension with 50 μM PP2 or DMSO solvent control (S) and plated on FN for 5 or 10 min. Immunoprecipitates were formed using either c-Raf antiserum, or matched control antiserum raised against the GAL4 transactivation domain, and kinase assays were performed with kinase-defective GST-MEK1 as described in Materials and methods (top). A reaction blank (lane B) confirms the absence of MEK kinase activity in the substrate and c-Raf antiserum, and a reaction lacking GST-MEK1 (lane 8) confirms that MEK1 is the phosphorylated substrate in these assays. Loading controls (bottom) demonstrate similar levels of c-Raf protein in the appropriate assays. (B) PAK1 activity was determined as described above for c-Raf activity (A) except cells were also plated for 5, 10, or 20 min and immunoprecipitates were formed using PAK1 antiserum. PAK1 activity was assessed by blotting the recombinant MEK1 substrate with the p-S298 antiserum (top). The PAK1 loading control is shown in the bottom panel. Lane B denotes a reaction blank in which PAK1 antibody was incubated with lysis buffer instead of lysate before the kinase assay.
Mentions: To examine whether PAK regulates adhesion-dependent MEK1 S298 phosphorylation in vivo, COS-1 or REF52 cells expressing kinase-defective PAK1 (or vector control) together with HA-tagged wild-type MEK1 were placed in suspension and replated on FN for the indicated times and S298 phosphorylation was assessed by Western blotting. Expression of kinase-defective PAK1 significantly reduced FN-stimulated S298 phosphorylation of exogenously expressed MEK1 (Fig. 4 A, not depicted), indicating that PAK activation is necessary for adhesion-dependent MEK1 S298 phosphorylation. To determine if PAK activity is sufficient to mediate MEK1 S298 phosphorylation in vivo, MEK1 S298 phosphorylation was examined in cells overexpressing activated PAK (T423E). REF52 cells expressing activated PAK together with wild-type MEK1 exhibited elevated MEK1 S298 phosphorylation whether in suspension or plated on FN (Fig. 4 B). Lastly, endogenous PAK1 immunoprecipitated from REF52 cells was activated by adhesion to FN (see Fig. 8 B), whereas endogenous PAK2 and 4 were not (not depicted). Together, these data indicate that PAK1 activity is both necessary and sufficient to stimulate MEK1 S298 phosphorylation and that PAK1-mediated phosphorylation of MEK1 on S298 is regulated by cell adhesion.

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