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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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Nuclear accumulation of ERK is impaired in nonadherent cells and by disruption of the actin cytoskeleton. Tet-Mek*-3T3 cells were serum starved and stimulated with 10% FCS in the absence of tetracycline for 6–9 h. The localization of active MEK and ERK was compared in adherent (Adh) and nonadherent (Sus) cells or adherent untreated (Adh) vs. CCD-treated (Adh/CCD) monolayers via confocal microscopy. Bottom panels are of (A) overlays of the MEK and ERK images or (B) images showing DAPI staining of nuclei. Scale bars represent either a 10 or 5 micron distance, as indicated. (C and D) tet-MEK*-3T3 cells were transfected with FLAG–Elk-1 as before. Cells were serum starved overnight, after which in some populations tetracycline was removed from the media to induce expression of active MEK. Cells were detached and either maintained in suspension (Sus) or replated onto fibronectin (Fn) and lysed 6 h after induction. (C) Levels of MEK expression and activation of ERK determined by Western blotting (WB) of whole cell lysates. (D) FLAG–Elk-1 was immunoprecipitated (IP) and analyzed by Western blotting for phosphorylated and total levels of Elk-1.
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Figure 5: Nuclear accumulation of ERK is impaired in nonadherent cells and by disruption of the actin cytoskeleton. Tet-Mek*-3T3 cells were serum starved and stimulated with 10% FCS in the absence of tetracycline for 6–9 h. The localization of active MEK and ERK was compared in adherent (Adh) and nonadherent (Sus) cells or adherent untreated (Adh) vs. CCD-treated (Adh/CCD) monolayers via confocal microscopy. Bottom panels are of (A) overlays of the MEK and ERK images or (B) images showing DAPI staining of nuclei. Scale bars represent either a 10 or 5 micron distance, as indicated. (C and D) tet-MEK*-3T3 cells were transfected with FLAG–Elk-1 as before. Cells were serum starved overnight, after which in some populations tetracycline was removed from the media to induce expression of active MEK. Cells were detached and either maintained in suspension (Sus) or replated onto fibronectin (Fn) and lysed 6 h after induction. (C) Levels of MEK expression and activation of ERK determined by Western blotting (WB) of whole cell lysates. (D) FLAG–Elk-1 was immunoprecipitated (IP) and analyzed by Western blotting for phosphorylated and total levels of Elk-1.

Mentions: The above findings suggested that ERK might be unable to redistribute properly to the nucleus upon activation in nonadherent conditions. To investigate this possibility, we used an NIH 3T3 cell line that expresses active MEK1 (tet-MEK*-3T3) under inducible control. Upon induction of active MEK in these cells, ERK is activated similarly under adherent and suspension conditions (Roovers et al. 1999). We used this system to analyze the role of adhesion upon the localization of ERK, after induction of active MEK. By confocal analysis, we determined that in adherent tet-MEK*-3T3 cells, ERK was localized primarily in the nuclear compartment, whereas MEK was present in the cytoplasm (Fig. 5 A, left). In contrast, in nonadherent cells, ERK extensively colocalized with MEK in the cytoplasmic compartment (Fig. 5 A, right). Higher magnification images confirmed that ERK was inefficiently translocated to the nucleus in nonadherent conditions (Fig. 5 B). Consistent with a role for integrin-associated actin structures playing an important role, ERK was poorly localized to the nucleus in adherent tet-MEK*-3T3 cells treated with CCD (Fig. 5 B). Quantification of >50 MEK-positive cells revealed that nuclear ERK staining was predominant in 75% of adherent cells, but only in 20 and 35% of cells treated with CCD and incubated in suspension, respectively. Moreover, some suspended and CCD-treated cells showed diffuse, rather than cytoplasm-specific, ERK staining. Similar observations on the immunolocalization of ERK were found in cells transiently expressing 22W Raf, in that ERK poorly distributed to the nucleus in nonadherent conditions (data not shown). The confocal analysis agrees well with our Elk-1 results which indicate that adhesion-dependent translocation of ERK in NIH 3T3 cells 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)

Nuclear accumulation of ERK is impaired in nonadherent cells and by disruption of the actin cytoskeleton. Tet-Mek*-3T3 cells were serum starved and stimulated with 10% FCS in the absence of tetracycline for 6–9 h. The localization of active MEK and ERK was compared in adherent (Adh) and nonadherent (Sus) cells or adherent untreated (Adh) vs. CCD-treated (Adh/CCD) monolayers via confocal microscopy. Bottom panels are of (A) overlays of the MEK and ERK images or (B) images showing DAPI staining of nuclei. Scale bars represent either a 10 or 5 micron distance, as indicated. (C and D) tet-MEK*-3T3 cells were transfected with FLAG–Elk-1 as before. Cells were serum starved overnight, after which in some populations tetracycline was removed from the media to induce expression of active MEK. Cells were detached and either maintained in suspension (Sus) or replated onto fibronectin (Fn) and lysed 6 h after induction. (C) Levels of MEK expression and activation of ERK determined by Western blotting (WB) of whole cell lysates. (D) FLAG–Elk-1 was immunoprecipitated (IP) and analyzed by Western blotting for phosphorylated and total levels of Elk-1.
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Figure 5: Nuclear accumulation of ERK is impaired in nonadherent cells and by disruption of the actin cytoskeleton. Tet-Mek*-3T3 cells were serum starved and stimulated with 10% FCS in the absence of tetracycline for 6–9 h. The localization of active MEK and ERK was compared in adherent (Adh) and nonadherent (Sus) cells or adherent untreated (Adh) vs. CCD-treated (Adh/CCD) monolayers via confocal microscopy. Bottom panels are of (A) overlays of the MEK and ERK images or (B) images showing DAPI staining of nuclei. Scale bars represent either a 10 or 5 micron distance, as indicated. (C and D) tet-MEK*-3T3 cells were transfected with FLAG–Elk-1 as before. Cells were serum starved overnight, after which in some populations tetracycline was removed from the media to induce expression of active MEK. Cells were detached and either maintained in suspension (Sus) or replated onto fibronectin (Fn) and lysed 6 h after induction. (C) Levels of MEK expression and activation of ERK determined by Western blotting (WB) of whole cell lysates. (D) FLAG–Elk-1 was immunoprecipitated (IP) and analyzed by Western blotting for phosphorylated and total levels of Elk-1.
Mentions: The above findings suggested that ERK might be unable to redistribute properly to the nucleus upon activation in nonadherent conditions. To investigate this possibility, we used an NIH 3T3 cell line that expresses active MEK1 (tet-MEK*-3T3) under inducible control. Upon induction of active MEK in these cells, ERK is activated similarly under adherent and suspension conditions (Roovers et al. 1999). We used this system to analyze the role of adhesion upon the localization of ERK, after induction of active MEK. By confocal analysis, we determined that in adherent tet-MEK*-3T3 cells, ERK was localized primarily in the nuclear compartment, whereas MEK was present in the cytoplasm (Fig. 5 A, left). In contrast, in nonadherent cells, ERK extensively colocalized with MEK in the cytoplasmic compartment (Fig. 5 A, right). Higher magnification images confirmed that ERK was inefficiently translocated to the nucleus in nonadherent conditions (Fig. 5 B). Consistent with a role for integrin-associated actin structures playing an important role, ERK was poorly localized to the nucleus in adherent tet-MEK*-3T3 cells treated with CCD (Fig. 5 B). Quantification of >50 MEK-positive cells revealed that nuclear ERK staining was predominant in 75% of adherent cells, but only in 20 and 35% of cells treated with CCD and incubated in suspension, respectively. Moreover, some suspended and CCD-treated cells showed diffuse, rather than cytoplasm-specific, ERK staining. Similar observations on the immunolocalization of ERK were found in cells transiently expressing 22W Raf, in that ERK poorly distributed to the nucleus in nonadherent conditions (data not shown). The confocal analysis agrees well with our Elk-1 results which indicate that adhesion-dependent translocation of ERK in NIH 3T3 cells 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