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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 MEK1 S298 phosphorylation promotes maximal MEK1 activation in response to growth factor stimulation. (A) REF52 cells were suspended for 90 min and either stimulated for 30 min with EGF and IGF-1 in suspension, replated on FN for 30 min, or stimulated with EGF and IGF-1 while they attached to FN for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (B) REF52 cells were placed in suspension for 90 min and either stimulated in suspension for 30 min with the indicated concentrations of EGF or replated on FN in the presence of the indicated concentrations of EGF for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (C) Cells were treated as in A, and whole cell lysates were blotted with p-S218/222 MEK or MEK1 antisera. (D) REF52 cells were transiently transfected with HA-MEK1 (lanes 1–6) or HA-MEK1 S298A (lanes 7–12). Cells were suspended in serum-free media for 90 min and either kept unstimulated in suspension (lanes 1 and 7), stimulated in suspension with EGF for 20 min (lanes 2 and 8), allowed to adhere to FN (lanes 3 and 9) or allowed to attach to FN while stimulated with EGF for the indicated times (lanes 4–6 and 10–12). Anti-HA immunoprecipitates were formed and blotted with p-S218/222 MEK1, p-S298MEK1 or HA antisera. Densitometry and normalization to the loading controls revealed that S218/222 phosphorylation of MEK1 S298A was ∼50% that seen in the wild-type protein.
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fig10: Adhesion-dependent MEK1 S298 phosphorylation promotes maximal MEK1 activation in response to growth factor stimulation. (A) REF52 cells were suspended for 90 min and either stimulated for 30 min with EGF and IGF-1 in suspension, replated on FN for 30 min, or stimulated with EGF and IGF-1 while they attached to FN for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (B) REF52 cells were placed in suspension for 90 min and either stimulated in suspension for 30 min with the indicated concentrations of EGF or replated on FN in the presence of the indicated concentrations of EGF for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (C) Cells were treated as in A, and whole cell lysates were blotted with p-S218/222 MEK or MEK1 antisera. (D) REF52 cells were transiently transfected with HA-MEK1 (lanes 1–6) or HA-MEK1 S298A (lanes 7–12). Cells were suspended in serum-free media for 90 min and either kept unstimulated in suspension (lanes 1 and 7), stimulated in suspension with EGF for 20 min (lanes 2 and 8), allowed to adhere to FN (lanes 3 and 9) or allowed to attach to FN while stimulated with EGF for the indicated times (lanes 4–6 and 10–12). Anti-HA immunoprecipitates were formed and blotted with p-S218/222 MEK1, p-S298MEK1 or HA antisera. Densitometry and normalization to the loading controls revealed that S218/222 phosphorylation of MEK1 S298A was ∼50% that seen in the wild-type protein.

Mentions: ECM adhesion has been reported to influence serum growth factor–stimulated MAPK activation at the level of Raf or MEK (Lin et al., 1997; Renshaw et al., 1997). Indeed, REF52 cells suspended for 90 min and stimulated with growth factors or replated on FN for 30 min resulted in a modest induction of MAPK phosphorylation (Fig. 10 A). Stimulating fibroblasts with growth factors while they adhere to FN for 30 min resulted in an enhanced activation of MAPK phosphorylation (Fig. 10, A and B) as reported previously (Roovers et al., 1999). The ability of FN to amplify growth factor–stimulated MAPK activity was most evident at lower, more physiologically relevant concentrations of EGF (0.125 ng/ml; Fig. 10 B, lanes 9 and 10), whereas higher concentrations of EGF (10 ng/ml) overcame the adhesion requirement to efficiently activate MAPK (Fig. 10 B, lanes 1 and 2). In addition, MEK1 activation was maximally activated by stimulation with both growth factors and adhesion to FN (Fig. 10 C). To determine whether MEK1 S298 phosphorylation plays a role in MEK1 activation in response to growth factor stimulation, HA-tagged wild-type MEK1 or MEK1 S298A was immunoprecipitated from REF52 cells that were serum-starved, suspended, and either stimulated with EGF in suspension, replated onto FN, or stimulated and replated. In the absence of adhesion, growth factor stimulation of REF52 cells resulted in equivalent S218/S222 phosphorylation of exogenously expressed wild-type MEK1 or MEK1 S298A (Fig. 10 D, lanes 2 and 8). However, growth factor stimulation of adherent cells expressing MEK1 S298A resulted in ∼50% less activation of MEK S218/S222 compared with cells expressing wild-type MEK1 (Fig. 10 D, lanes 4–6 and 10–12). Together, these results indicate that adhesion-dependent MEK1 S298 phosphorylation regulates, at least in part, the maximal activation of MEK1 by serum growth factors.


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 MEK1 S298 phosphorylation promotes maximal MEK1 activation in response to growth factor stimulation. (A) REF52 cells were suspended for 90 min and either stimulated for 30 min with EGF and IGF-1 in suspension, replated on FN for 30 min, or stimulated with EGF and IGF-1 while they attached to FN for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (B) REF52 cells were placed in suspension for 90 min and either stimulated in suspension for 30 min with the indicated concentrations of EGF or replated on FN in the presence of the indicated concentrations of EGF for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (C) Cells were treated as in A, and whole cell lysates were blotted with p-S218/222 MEK or MEK1 antisera. (D) REF52 cells were transiently transfected with HA-MEK1 (lanes 1–6) or HA-MEK1 S298A (lanes 7–12). Cells were suspended in serum-free media for 90 min and either kept unstimulated in suspension (lanes 1 and 7), stimulated in suspension with EGF for 20 min (lanes 2 and 8), allowed to adhere to FN (lanes 3 and 9) or allowed to attach to FN while stimulated with EGF for the indicated times (lanes 4–6 and 10–12). Anti-HA immunoprecipitates were formed and blotted with p-S218/222 MEK1, p-S298MEK1 or HA antisera. Densitometry and normalization to the loading controls revealed that S218/222 phosphorylation of MEK1 S298A was ∼50% that seen in the wild-type protein.
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fig10: Adhesion-dependent MEK1 S298 phosphorylation promotes maximal MEK1 activation in response to growth factor stimulation. (A) REF52 cells were suspended for 90 min and either stimulated for 30 min with EGF and IGF-1 in suspension, replated on FN for 30 min, or stimulated with EGF and IGF-1 while they attached to FN for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (B) REF52 cells were placed in suspension for 90 min and either stimulated in suspension for 30 min with the indicated concentrations of EGF or replated on FN in the presence of the indicated concentrations of EGF for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (C) Cells were treated as in A, and whole cell lysates were blotted with p-S218/222 MEK or MEK1 antisera. (D) REF52 cells were transiently transfected with HA-MEK1 (lanes 1–6) or HA-MEK1 S298A (lanes 7–12). Cells were suspended in serum-free media for 90 min and either kept unstimulated in suspension (lanes 1 and 7), stimulated in suspension with EGF for 20 min (lanes 2 and 8), allowed to adhere to FN (lanes 3 and 9) or allowed to attach to FN while stimulated with EGF for the indicated times (lanes 4–6 and 10–12). Anti-HA immunoprecipitates were formed and blotted with p-S218/222 MEK1, p-S298MEK1 or HA antisera. Densitometry and normalization to the loading controls revealed that S218/222 phosphorylation of MEK1 S298A was ∼50% that seen in the wild-type protein.
Mentions: ECM adhesion has been reported to influence serum growth factor–stimulated MAPK activation at the level of Raf or MEK (Lin et al., 1997; Renshaw et al., 1997). Indeed, REF52 cells suspended for 90 min and stimulated with growth factors or replated on FN for 30 min resulted in a modest induction of MAPK phosphorylation (Fig. 10 A). Stimulating fibroblasts with growth factors while they adhere to FN for 30 min resulted in an enhanced activation of MAPK phosphorylation (Fig. 10, A and B) as reported previously (Roovers et al., 1999). The ability of FN to amplify growth factor–stimulated MAPK activity was most evident at lower, more physiologically relevant concentrations of EGF (0.125 ng/ml; Fig. 10 B, lanes 9 and 10), whereas higher concentrations of EGF (10 ng/ml) overcame the adhesion requirement to efficiently activate MAPK (Fig. 10 B, lanes 1 and 2). In addition, MEK1 activation was maximally activated by stimulation with both growth factors and adhesion to FN (Fig. 10 C). To determine whether MEK1 S298 phosphorylation plays a role in MEK1 activation in response to growth factor stimulation, HA-tagged wild-type MEK1 or MEK1 S298A was immunoprecipitated from REF52 cells that were serum-starved, suspended, and either stimulated with EGF in suspension, replated onto FN, or stimulated and replated. In the absence of adhesion, growth factor stimulation of REF52 cells resulted in equivalent S218/S222 phosphorylation of exogenously expressed wild-type MEK1 or MEK1 S298A (Fig. 10 D, lanes 2 and 8). However, growth factor stimulation of adherent cells expressing MEK1 S298A resulted in ∼50% less activation of MEK S218/S222 compared with cells expressing wild-type MEK1 (Fig. 10 D, lanes 4–6 and 10–12). Together, these results indicate that adhesion-dependent MEK1 S298 phosphorylation regulates, at least in part, the maximal activation of MEK1 by serum growth factors.

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