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Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1.

Aplin AE, Stewart SA, Assoian RK, Juliano RL - J. Cell Biol. (2001)

Bottom Line: Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells.Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD.These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. aaplin@med.unc.edu

ABSTRACT
Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.

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Disruption of the actin cytoskeleton, but not the microtubule network, inhibits the ability of activated ERK to phosphorylate Elk-1. NIH 3T3 cells were transfected either with vector (Vec) or 22W Raf and either FLAG-Elk-1 (A and C) or HA-ERK1 (B). In A and B, cells were treated accordingly with 2 μM CCD throughout adhesion to fibronectin-coated plates (Fn). In C, serum-starved cells were treated with 50 μM colchicine (Colc), as indicated, before replating either on fibronectin-coated plates (Fn) or maintained in suspension (Sus) for 3 h. FLAG-Elk-1 immunoprecipitates (IP) were analyzed by Western blotting (WB) for levels of serine 383 phosphorylated and total Elk-1 (A and C). Additionally, in A (bottom) endogenous FAK was immunoprecipitated from cell lysates and blotted for tyrosine phosphorylation (PY) and total levels of FAK. In B, HA-ERK was immunoprecipitated and activity measured by in vitro kinase assay. Shown are representatives of at least three independent experiments with equivalent results.
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Figure 4: Disruption of the actin cytoskeleton, but not the microtubule network, inhibits the ability of activated ERK to phosphorylate Elk-1. NIH 3T3 cells were transfected either with vector (Vec) or 22W Raf and either FLAG-Elk-1 (A and C) or HA-ERK1 (B). In A and B, cells were treated accordingly with 2 μM CCD throughout adhesion to fibronectin-coated plates (Fn). In C, serum-starved cells were treated with 50 μM colchicine (Colc), as indicated, before replating either on fibronectin-coated plates (Fn) or maintained in suspension (Sus) for 3 h. FLAG-Elk-1 immunoprecipitates (IP) were analyzed by Western blotting (WB) for levels of serine 383 phosphorylated and total Elk-1 (A and C). Additionally, in A (bottom) endogenous FAK was immunoprecipitated from cell lysates and blotted for tyrosine phosphorylation (PY) and total levels of FAK. In B, HA-ERK was immunoprecipitated and activity measured by in vitro kinase assay. Shown are representatives of at least three independent experiments with equivalent results.

Mentions: Integrin signaling events are typically dependent on an intact actin cytoskeleton. We used the actin depolymerizing agent, CCD, that caps the ends of growing actin fibers and inhibits integrin-mediated tyrosine phosphorylation of focal adhesion proteins (Burridge et al. 1992). Treatment of 22W Raf–expressing cells with CCD significantly reduced ERK phosphorylation of Elk-1 in adherent cells (Fig. 4 A, top). Under these conditions, cells remained round but firmly attached when viewed by microscopy, and FAK phosphotyrosine levels were dramatically reduced (Fig. 4 A, bottom). Overall, in these transient transfection experiments, the CCD effect was not quite as dramatic as the inhibition of Elk-1 phosphorylation in suspension. In vitro kinase assays demonstrated that ERK activation by 22W Raf was not inhibited by treatment with CCD (Fig. 4 B). In contrast, treatment of 22W Raf–expressing cells with the microtubule-disrupting agent, colchicine, did not inhibit Elk-1 phosphorylation at serine 383 (Fig. 4 C). Thus, the ability of ERK, once activated, to phosphorylate Elk-1 is dependent on an intact actin cytoskeleton, but not the microtubule network.


Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1.

Aplin AE, Stewart SA, Assoian RK, Juliano RL - J. Cell Biol. (2001)

Disruption of the actin cytoskeleton, but not the microtubule network, inhibits the ability of activated ERK to phosphorylate Elk-1. NIH 3T3 cells were transfected either with vector (Vec) or 22W Raf and either FLAG-Elk-1 (A and C) or HA-ERK1 (B). In A and B, cells were treated accordingly with 2 μM CCD throughout adhesion to fibronectin-coated plates (Fn). In C, serum-starved cells were treated with 50 μM colchicine (Colc), as indicated, before replating either on fibronectin-coated plates (Fn) or maintained in suspension (Sus) for 3 h. FLAG-Elk-1 immunoprecipitates (IP) were analyzed by Western blotting (WB) for levels of serine 383 phosphorylated and total Elk-1 (A and C). Additionally, in A (bottom) endogenous FAK was immunoprecipitated from cell lysates and blotted for tyrosine phosphorylation (PY) and total levels of FAK. In B, HA-ERK was immunoprecipitated and activity measured by in vitro kinase assay. Shown are representatives of at least three independent experiments with equivalent results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169466&req=5

Figure 4: Disruption of the actin cytoskeleton, but not the microtubule network, inhibits the ability of activated ERK to phosphorylate Elk-1. NIH 3T3 cells were transfected either with vector (Vec) or 22W Raf and either FLAG-Elk-1 (A and C) or HA-ERK1 (B). In A and B, cells were treated accordingly with 2 μM CCD throughout adhesion to fibronectin-coated plates (Fn). In C, serum-starved cells were treated with 50 μM colchicine (Colc), as indicated, before replating either on fibronectin-coated plates (Fn) or maintained in suspension (Sus) for 3 h. FLAG-Elk-1 immunoprecipitates (IP) were analyzed by Western blotting (WB) for levels of serine 383 phosphorylated and total Elk-1 (A and C). Additionally, in A (bottom) endogenous FAK was immunoprecipitated from cell lysates and blotted for tyrosine phosphorylation (PY) and total levels of FAK. In B, HA-ERK was immunoprecipitated and activity measured by in vitro kinase assay. Shown are representatives of at least three independent experiments with equivalent results.
Mentions: Integrin signaling events are typically dependent on an intact actin cytoskeleton. We used the actin depolymerizing agent, CCD, that caps the ends of growing actin fibers and inhibits integrin-mediated tyrosine phosphorylation of focal adhesion proteins (Burridge et al. 1992). Treatment of 22W Raf–expressing cells with CCD significantly reduced ERK phosphorylation of Elk-1 in adherent cells (Fig. 4 A, top). Under these conditions, cells remained round but firmly attached when viewed by microscopy, and FAK phosphotyrosine levels were dramatically reduced (Fig. 4 A, bottom). Overall, in these transient transfection experiments, the CCD effect was not quite as dramatic as the inhibition of Elk-1 phosphorylation in suspension. In vitro kinase assays demonstrated that ERK activation by 22W Raf was not inhibited by treatment with CCD (Fig. 4 B). In contrast, treatment of 22W Raf–expressing cells with the microtubule-disrupting agent, colchicine, did not inhibit Elk-1 phosphorylation at serine 383 (Fig. 4 C). Thus, the ability of ERK, once activated, to phosphorylate Elk-1 is dependent on an intact actin cytoskeleton, but not the microtubule network.

Bottom Line: Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells.Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD.These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. aaplin@med.unc.edu

ABSTRACT
Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.

Show MeSH
Related in: MedlinePlus