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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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Adhesion to fibronectin and EGF collaborate to provide efficient phosphorylation of the Elk-1 transcription factor. NIH 3T3 cells were transfected with either pCMV5 (Vector) or pCMV5-FLAG-Elk-1. In A, 1 μg of green fluorescent protein (GFP) was included in the transfections to identify transfected cells. After 48 h, transfected cells were serum starved before being replated in DMEM/BSA on fibronectin-coated coverslips (A) or maintained in suspension (Sus) or replated on fibronectin (Fn)-coated dishes for a further 3 h (B). (A) Localization of Elk-1 was determined by immunofluorescence with an Elk-1 antibody and TRITC-conjugated anti–rabbit secondary antibody. The scale bar depicts a 10 micron distance. (B) After the 3-h incubation, cells were treated with 20 ng/ml EGF for 15 min as indicated. Ectopically expressed Elk-1 was immunoprecipitated (IP) from cell lysates from each condition with an M2 FLAG epitope antibody. Immunoprecipitates were analyzed by Western blotting (WB) with antibodies to determine phosphorylated and total Elk-1 levels.
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Figure 1: Adhesion to fibronectin and EGF collaborate to provide efficient phosphorylation of the Elk-1 transcription factor. NIH 3T3 cells were transfected with either pCMV5 (Vector) or pCMV5-FLAG-Elk-1. In A, 1 μg of green fluorescent protein (GFP) was included in the transfections to identify transfected cells. After 48 h, transfected cells were serum starved before being replated in DMEM/BSA on fibronectin-coated coverslips (A) or maintained in suspension (Sus) or replated on fibronectin (Fn)-coated dishes for a further 3 h (B). (A) Localization of Elk-1 was determined by immunofluorescence with an Elk-1 antibody and TRITC-conjugated anti–rabbit secondary antibody. The scale bar depicts a 10 micron distance. (B) After the 3-h incubation, cells were treated with 20 ng/ml EGF for 15 min as indicated. Ectopically expressed Elk-1 was immunoprecipitated (IP) from cell lysates from each condition with an M2 FLAG epitope antibody. Immunoprecipitates were analyzed by Western blotting (WB) with antibodies to determine phosphorylated and total Elk-1 levels.

Mentions: Activation of ERK by growth factors in human and mouse fibroblasts is dependent on the state of adhesion (Miyamoto et al. 1996; Lin et al. 1997; Renshaw et al. 1997; Aplin and Juliano 1999). The transcription factor Elk-1 is a substrate for ERK, and phosphorylation at several COOH-terminal sites, including serine 383, is critical for its transcriptional potential (Marais et al. 1993; Whitmarsh et al. 1995). We initially determined whether Elk-1 displays anchorage-dependent phosphorylation in response to growth factors. NIH 3T3 fibroblasts express low levels of endogenous Elk-1, hence we used transient transfection of a FLAG-tagged version of Elk-1 which localized to the nucleus as determined by immunofluorescence. Identical results were obtained with antibodies to Elk-1 (Fig. 1 A) and the FLAG epitope (data not shown). The phosphorylation status of Elk-1 was monitored by immunoprecipitation followed by Western blotting with a serine 383 phosphorylation state–dependent antibody. When transfected cells adherent to fibronectin were stimulated with EGF, Elk-1 was robustly phosphorylated at serine 383 (Fig. 1 B). In contrast, Elk-1 was weakly phosphorylated upon EGF treatment in nonadherent cells. Thus, Elk-1 phosphorylation is adhesion dependent in response to growth factors in a manner that closely correlates with adhesion effects on ERK activation (Lin et al. 1997; Aplin and Juliano 1999).


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

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

Adhesion to fibronectin and EGF collaborate to provide efficient phosphorylation of the Elk-1 transcription factor. NIH 3T3 cells were transfected with either pCMV5 (Vector) or pCMV5-FLAG-Elk-1. In A, 1 μg of green fluorescent protein (GFP) was included in the transfections to identify transfected cells. After 48 h, transfected cells were serum starved before being replated in DMEM/BSA on fibronectin-coated coverslips (A) or maintained in suspension (Sus) or replated on fibronectin (Fn)-coated dishes for a further 3 h (B). (A) Localization of Elk-1 was determined by immunofluorescence with an Elk-1 antibody and TRITC-conjugated anti–rabbit secondary antibody. The scale bar depicts a 10 micron distance. (B) After the 3-h incubation, cells were treated with 20 ng/ml EGF for 15 min as indicated. Ectopically expressed Elk-1 was immunoprecipitated (IP) from cell lysates from each condition with an M2 FLAG epitope antibody. Immunoprecipitates were analyzed by Western blotting (WB) with antibodies to determine phosphorylated and total Elk-1 levels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169466&req=5

Figure 1: Adhesion to fibronectin and EGF collaborate to provide efficient phosphorylation of the Elk-1 transcription factor. NIH 3T3 cells were transfected with either pCMV5 (Vector) or pCMV5-FLAG-Elk-1. In A, 1 μg of green fluorescent protein (GFP) was included in the transfections to identify transfected cells. After 48 h, transfected cells were serum starved before being replated in DMEM/BSA on fibronectin-coated coverslips (A) or maintained in suspension (Sus) or replated on fibronectin (Fn)-coated dishes for a further 3 h (B). (A) Localization of Elk-1 was determined by immunofluorescence with an Elk-1 antibody and TRITC-conjugated anti–rabbit secondary antibody. The scale bar depicts a 10 micron distance. (B) After the 3-h incubation, cells were treated with 20 ng/ml EGF for 15 min as indicated. Ectopically expressed Elk-1 was immunoprecipitated (IP) from cell lysates from each condition with an M2 FLAG epitope antibody. Immunoprecipitates were analyzed by Western blotting (WB) with antibodies to determine phosphorylated and total Elk-1 levels.
Mentions: Activation of ERK by growth factors in human and mouse fibroblasts is dependent on the state of adhesion (Miyamoto et al. 1996; Lin et al. 1997; Renshaw et al. 1997; Aplin and Juliano 1999). The transcription factor Elk-1 is a substrate for ERK, and phosphorylation at several COOH-terminal sites, including serine 383, is critical for its transcriptional potential (Marais et al. 1993; Whitmarsh et al. 1995). We initially determined whether Elk-1 displays anchorage-dependent phosphorylation in response to growth factors. NIH 3T3 fibroblasts express low levels of endogenous Elk-1, hence we used transient transfection of a FLAG-tagged version of Elk-1 which localized to the nucleus as determined by immunofluorescence. Identical results were obtained with antibodies to Elk-1 (Fig. 1 A) and the FLAG epitope (data not shown). The phosphorylation status of Elk-1 was monitored by immunoprecipitation followed by Western blotting with a serine 383 phosphorylation state–dependent antibody. When transfected cells adherent to fibronectin were stimulated with EGF, Elk-1 was robustly phosphorylated at serine 383 (Fig. 1 B). In contrast, Elk-1 was weakly phosphorylated upon EGF treatment in nonadherent cells. Thus, Elk-1 phosphorylation is adhesion dependent in response to growth factors in a manner that closely correlates with adhesion effects on ERK activation (Lin et al. 1997; Aplin and Juliano 1999).

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