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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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Phosphorylation and transcriptional activity of Elk-1 mediated by activated ERK are impaired in nonadherent cells. NIH 3T3 cells were transfected with FLAG–Elk-1 and either vector (Vec), 22W Raf (A), or MEK1-ΔED (B). Serum-starved cells were either replated 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. Shown are representatives of at least three independent experiments with equivalent results. In C, cells were transfected with GAL4–Elk-1, pFR-luc reporter, and either vector or 22W Raf. After a brief serum starvation, cells were replated as above on fibronectin-coated plates (Fn) or maintained in suspension (Sus). The increase in GAL4-Elk-1 transactivation of pFR-luc during a 4-h time period was determined by assaying for firefly luciferase activity. For each experiment, three separate samples were assayed for each condition and all readings were normalized to the activity of Renilla luciferase under the control of a constitutively active CMV promoter (pRL-CMV-luc). The enhanced GAL4–Elk-1–driven luciferase activity in 22W Raf–expressing cells in adherent compared with suspended cells is statistically significant (*P < 0.05).
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Figure 3: Phosphorylation and transcriptional activity of Elk-1 mediated by activated ERK are impaired in nonadherent cells. NIH 3T3 cells were transfected with FLAG–Elk-1 and either vector (Vec), 22W Raf (A), or MEK1-ΔED (B). Serum-starved cells were either replated 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. Shown are representatives of at least three independent experiments with equivalent results. In C, cells were transfected with GAL4–Elk-1, pFR-luc reporter, and either vector or 22W Raf. After a brief serum starvation, cells were replated as above on fibronectin-coated plates (Fn) or maintained in suspension (Sus). The increase in GAL4-Elk-1 transactivation of pFR-luc during a 4-h time period was determined by assaying for firefly luciferase activity. For each experiment, three separate samples were assayed for each condition and all readings were normalized to the activity of Renilla luciferase under the control of a constitutively active CMV promoter (pRL-CMV-luc). The enhanced GAL4–Elk-1–driven luciferase activity in 22W Raf–expressing cells in adherent compared with suspended cells is statistically significant (*P < 0.05).

Mentions: To examine whether active ERK was able to phosphorylate Elk-1 at serine 383 in the absence of integrin engagement, we cotransfected cells with Elk-1 and either 22W Raf or empty vector. As before, control transfected cells exhibited little phosphorylation of Elk-1 when either held in suspension or readhered to fibronectin for 3 h in the absence of growth factors (Fig. 3 A). Expression of 22W Raf stimulated ERK phosphorylation of Elk-1 at serine 383 in adherent cells, but this effect was markedly reduced under suspension conditions (Fig. 3 A). The levels of Elk-1 and 22W Raf were comparable between the suspension and fibronectin conditions (Fig. 3 A, and latter data not shown). Similarly, expression of MEK1-ΔED resulted in efficient phosphorylation of serine 383 in Elk-1 when cells were adherent to fibronectin but not under suspension conditions (Fig. 3 B). In both the 22W Raf and MEK1-ΔED experiments, there remained a small but noticeable increase in the phosphorylation of Elk-1 above control conditions in suspended cells. Thus, the adhesion effect on ERK-mediated phosphorylation of Elk-1 is potent but not complete.


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

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

Phosphorylation and transcriptional activity of Elk-1 mediated by activated ERK are impaired in nonadherent cells. NIH 3T3 cells were transfected with FLAG–Elk-1 and either vector (Vec), 22W Raf (A), or MEK1-ΔED (B). Serum-starved cells were either replated 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. Shown are representatives of at least three independent experiments with equivalent results. In C, cells were transfected with GAL4–Elk-1, pFR-luc reporter, and either vector or 22W Raf. After a brief serum starvation, cells were replated as above on fibronectin-coated plates (Fn) or maintained in suspension (Sus). The increase in GAL4-Elk-1 transactivation of pFR-luc during a 4-h time period was determined by assaying for firefly luciferase activity. For each experiment, three separate samples were assayed for each condition and all readings were normalized to the activity of Renilla luciferase under the control of a constitutively active CMV promoter (pRL-CMV-luc). The enhanced GAL4–Elk-1–driven luciferase activity in 22W Raf–expressing cells in adherent compared with suspended cells is statistically significant (*P < 0.05).
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Related In: Results  -  Collection

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Figure 3: Phosphorylation and transcriptional activity of Elk-1 mediated by activated ERK are impaired in nonadherent cells. NIH 3T3 cells were transfected with FLAG–Elk-1 and either vector (Vec), 22W Raf (A), or MEK1-ΔED (B). Serum-starved cells were either replated 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. Shown are representatives of at least three independent experiments with equivalent results. In C, cells were transfected with GAL4–Elk-1, pFR-luc reporter, and either vector or 22W Raf. After a brief serum starvation, cells were replated as above on fibronectin-coated plates (Fn) or maintained in suspension (Sus). The increase in GAL4-Elk-1 transactivation of pFR-luc during a 4-h time period was determined by assaying for firefly luciferase activity. For each experiment, three separate samples were assayed for each condition and all readings were normalized to the activity of Renilla luciferase under the control of a constitutively active CMV promoter (pRL-CMV-luc). The enhanced GAL4–Elk-1–driven luciferase activity in 22W Raf–expressing cells in adherent compared with suspended cells is statistically significant (*P < 0.05).
Mentions: To examine whether active ERK was able to phosphorylate Elk-1 at serine 383 in the absence of integrin engagement, we cotransfected cells with Elk-1 and either 22W Raf or empty vector. As before, control transfected cells exhibited little phosphorylation of Elk-1 when either held in suspension or readhered to fibronectin for 3 h in the absence of growth factors (Fig. 3 A). Expression of 22W Raf stimulated ERK phosphorylation of Elk-1 at serine 383 in adherent cells, but this effect was markedly reduced under suspension conditions (Fig. 3 A). The levels of Elk-1 and 22W Raf were comparable between the suspension and fibronectin conditions (Fig. 3 A, and latter data not shown). Similarly, expression of MEK1-ΔED resulted in efficient phosphorylation of serine 383 in Elk-1 when cells were adherent to fibronectin but not under suspension conditions (Fig. 3 B). In both the 22W Raf and MEK1-ΔED experiments, there remained a small but noticeable increase in the phosphorylation of Elk-1 above control conditions in suspended cells. Thus, the adhesion effect on ERK-mediated phosphorylation of Elk-1 is potent but not complete.

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