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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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Src inhibition alters PAK-mediated MEK phosphorylation. (A) REF52 cells were incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on 10 μg/ml FN for 5, 10, 20, or 40 min in the continued presence or absence of PP2. Whole cell lysates were blotted with p-S298MEK1, p-S218/S222MEK1, MEK1, p-MAPK, or ERK2 antisera. Densitometric analysis demonstrated that the intensity of p-S298MEK1 after a 5-min plating on FN in the presence of 50 μM PP2 was ∼28% the level of the DMSO-treated control when normalized to an MEK1 loading control. The observed decrease in p-MAPK levels in lane 9 was not reproducible. (B) REF52 cells were cotransfected with HA-tagged MEK1 constructs and PAK1 T423E or vector control, incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on FN for 20 min. Anti-HA immunoprecipitates were formed and blotted with pS218/S222 antiserum (top), HA antiserum (second [from top] panel), and subsequently with anti-pS298MEK1 (third [from top] panel). Western blotting of lysates with anti-myc antiserum confirmed expression of activated PAK1 (bottom).
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fig7: Src inhibition alters PAK-mediated MEK phosphorylation. (A) REF52 cells were incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on 10 μg/ml FN for 5, 10, 20, or 40 min in the continued presence or absence of PP2. Whole cell lysates were blotted with p-S298MEK1, p-S218/S222MEK1, MEK1, p-MAPK, or ERK2 antisera. Densitometric analysis demonstrated that the intensity of p-S298MEK1 after a 5-min plating on FN in the presence of 50 μM PP2 was ∼28% the level of the DMSO-treated control when normalized to an MEK1 loading control. The observed decrease in p-MAPK levels in lane 9 was not reproducible. (B) REF52 cells were cotransfected with HA-tagged MEK1 constructs and PAK1 T423E or vector control, incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on FN for 20 min. Anti-HA immunoprecipitates were formed and blotted with pS218/S222 antiserum (top), HA antiserum (second [from top] panel), and subsequently with anti-pS298MEK1 (third [from top] panel). Western blotting of lysates with anti-myc antiserum confirmed expression of activated PAK1 (bottom).

Mentions: Adhesion-mediated activation of FAK results in the stimulation of Src after binding to FAK phosphorylated on Y397 (Cobb et al., 1994; Schaller et al., 1994, 1999; Reiske et al., 1999). Because Y397F FAK was unable to restore MEK1 S298 phosphorylation, we assessed the role of Src in regulating adhesion-dependent MEK1 S298 phosphorylation using the Src family kinase inhibitor, PP2. REF52 cells were suspended in serum-free media in the presence or absence of PP2, and replated on FN in the continued presence or absence of PP2 (Fig. 7 A). PP2 treatment decreased the extent and delayed the time course of S298 phosphorylation after FN stimulation (Fig. 7 A), similar to the observations for FAK- cells (Fig. 6 A). MEK1 S298 phosphorylation in cells stimulated to adhere to FN for 5 min in the presence of PP2 was ∼28% the level of untreated cells when normalized to MEK1 loading control. Overexpression of activated PAK (PAK1 T423E) rescued the decrease in MEK1 S298 phosphorylation induced by PP2 treatment (Fig. 7 B). In addition, active PAK-stimulated MEK1 S218/S222 phosphorylation on wild-type MEK1 and to a lesser degree on MEK1 S298A. Finally, cells deficient for Src, Yes, and Fyn also showed decreased and delayed MEK1 S298 phosphorylation upon plating on FN (Fig. S2). These results indicate that Src or a Src family kinase is involved in regulating the pathway(s) leading to 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)

Src inhibition alters PAK-mediated MEK phosphorylation. (A) REF52 cells were incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on 10 μg/ml FN for 5, 10, 20, or 40 min in the continued presence or absence of PP2. Whole cell lysates were blotted with p-S298MEK1, p-S218/S222MEK1, MEK1, p-MAPK, or ERK2 antisera. Densitometric analysis demonstrated that the intensity of p-S298MEK1 after a 5-min plating on FN in the presence of 50 μM PP2 was ∼28% the level of the DMSO-treated control when normalized to an MEK1 loading control. The observed decrease in p-MAPK levels in lane 9 was not reproducible. (B) REF52 cells were cotransfected with HA-tagged MEK1 constructs and PAK1 T423E or vector control, incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on FN for 20 min. Anti-HA immunoprecipitates were formed and blotted with pS218/S222 antiserum (top), HA antiserum (second [from top] panel), and subsequently with anti-pS298MEK1 (third [from top] panel). Western blotting of lysates with anti-myc antiserum confirmed expression of activated PAK1 (bottom).
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fig7: Src inhibition alters PAK-mediated MEK phosphorylation. (A) REF52 cells were incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on 10 μg/ml FN for 5, 10, 20, or 40 min in the continued presence or absence of PP2. Whole cell lysates were blotted with p-S298MEK1, p-S218/S222MEK1, MEK1, p-MAPK, or ERK2 antisera. Densitometric analysis demonstrated that the intensity of p-S298MEK1 after a 5-min plating on FN in the presence of 50 μM PP2 was ∼28% the level of the DMSO-treated control when normalized to an MEK1 loading control. The observed decrease in p-MAPK levels in lane 9 was not reproducible. (B) REF52 cells were cotransfected with HA-tagged MEK1 constructs and PAK1 T423E or vector control, incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on FN for 20 min. Anti-HA immunoprecipitates were formed and blotted with pS218/S222 antiserum (top), HA antiserum (second [from top] panel), and subsequently with anti-pS298MEK1 (third [from top] panel). Western blotting of lysates with anti-myc antiserum confirmed expression of activated PAK1 (bottom).
Mentions: Adhesion-mediated activation of FAK results in the stimulation of Src after binding to FAK phosphorylated on Y397 (Cobb et al., 1994; Schaller et al., 1994, 1999; Reiske et al., 1999). Because Y397F FAK was unable to restore MEK1 S298 phosphorylation, we assessed the role of Src in regulating adhesion-dependent MEK1 S298 phosphorylation using the Src family kinase inhibitor, PP2. REF52 cells were suspended in serum-free media in the presence or absence of PP2, and replated on FN in the continued presence or absence of PP2 (Fig. 7 A). PP2 treatment decreased the extent and delayed the time course of S298 phosphorylation after FN stimulation (Fig. 7 A), similar to the observations for FAK- cells (Fig. 6 A). MEK1 S298 phosphorylation in cells stimulated to adhere to FN for 5 min in the presence of PP2 was ∼28% the level of untreated cells when normalized to MEK1 loading control. Overexpression of activated PAK (PAK1 T423E) rescued the decrease in MEK1 S298 phosphorylation induced by PP2 treatment (Fig. 7 B). In addition, active PAK-stimulated MEK1 S218/S222 phosphorylation on wild-type MEK1 and to a lesser degree on MEK1 S298A. Finally, cells deficient for Src, Yes, and Fyn also showed decreased and delayed MEK1 S298 phosphorylation upon plating on FN (Fig. S2). These results indicate that Src or a Src family kinase is involved in regulating the pathway(s) leading to 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