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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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Targeting of cyclin D1 to the nucleus in not affected by disruption of the actin cytoskeleton. Tet-cyclin D1-3T3 cells were G0-synchronized and stimulated with 10% FCS in the absence of tetracycline in monolayer in the absence and presence of CCD. The cells were fixed 6 h after stimulation, stained with anti-cyclin D1 antibody and DAPI nuclear stain, and analyzed via confocal microscopy. An overlay of the cyclin D1 and DAPI images is shown. The scale bars show a 10 micron distance. Adh, adherent.
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Figure 6: Targeting of cyclin D1 to the nucleus in not affected by disruption of the actin cytoskeleton. Tet-cyclin D1-3T3 cells were G0-synchronized and stimulated with 10% FCS in the absence of tetracycline in monolayer in the absence and presence of CCD. The cells were fixed 6 h after stimulation, stained with anti-cyclin D1 antibody and DAPI nuclear stain, and analyzed via confocal microscopy. An overlay of the cyclin D1 and DAPI images is shown. The scale bars show a 10 micron distance. Adh, adherent.

Mentions: Similar to ERK, cyclin D1 does not contain a consensus nuclear localization sequence; rather, it is localized to the nucleus via such sequences in the cyclin-dependent kinase inhibitors, p21cip1 and p27kip1 (LaBaer et al. 1997; Cheng et al. 1999). To examine the possibility that disruption of the actin cytoskeleton has a global effect on nucleocytoplasmic transport, we analyzed nuclear localization of cyclin D1 in CCD-treated cells. Confocal immunolocalization experiments showed that nuclear accumulation of ectopically expressed cyclin D1 in tet-cyclin D1-3T3 cells, was not altered upon disruption of the actin cytoskeleton as indicated by its colocalization with DAPI-stained nuclei (Fig. 6). Thus, nuclear accumulation of cyclin D1 protein via a consensus nuclear signal import mechanism is not dependent on an intact actin cytoskeleton.


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

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

Targeting of cyclin D1 to the nucleus in not affected by disruption of the actin cytoskeleton. Tet-cyclin D1-3T3 cells were G0-synchronized and stimulated with 10% FCS in the absence of tetracycline in monolayer in the absence and presence of CCD. The cells were fixed 6 h after stimulation, stained with anti-cyclin D1 antibody and DAPI nuclear stain, and analyzed via confocal microscopy. An overlay of the cyclin D1 and DAPI images is shown. The scale bars show a 10 micron distance. Adh, adherent.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169466&req=5

Figure 6: Targeting of cyclin D1 to the nucleus in not affected by disruption of the actin cytoskeleton. Tet-cyclin D1-3T3 cells were G0-synchronized and stimulated with 10% FCS in the absence of tetracycline in monolayer in the absence and presence of CCD. The cells were fixed 6 h after stimulation, stained with anti-cyclin D1 antibody and DAPI nuclear stain, and analyzed via confocal microscopy. An overlay of the cyclin D1 and DAPI images is shown. The scale bars show a 10 micron distance. Adh, adherent.
Mentions: Similar to ERK, cyclin D1 does not contain a consensus nuclear localization sequence; rather, it is localized to the nucleus via such sequences in the cyclin-dependent kinase inhibitors, p21cip1 and p27kip1 (LaBaer et al. 1997; Cheng et al. 1999). To examine the possibility that disruption of the actin cytoskeleton has a global effect on nucleocytoplasmic transport, we analyzed nuclear localization of cyclin D1 in CCD-treated cells. Confocal immunolocalization experiments showed that nuclear accumulation of ectopically expressed cyclin D1 in tet-cyclin D1-3T3 cells, was not altered upon disruption of the actin cytoskeleton as indicated by its colocalization with DAPI-stained nuclei (Fig. 6). Thus, nuclear accumulation of cyclin D1 protein via a consensus nuclear signal import mechanism is not dependent on an intact actin cytoskeleton.

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