Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1 - PubMed (original) (raw)

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

A E Aplin et al. J Cell Biol. 2001.

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.

PubMed Disclaimer

Figures

Figure 1

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.

Figure 1

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.

Figure 3

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).

Figure 3

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).

Figure 3

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).

Figure 2

Figure 2

Expression of active Raf or MEK in suspended cells is sufficient to activate ERK activity. (A) NIH 3T3 cells transfected either with pcDNA3 vector (Vec) or pcDNA3-22W Raf were analyzed by Western blotting (WB) with the anti-Raf COOH-terminal (C-term) antibody, C12, 48 h after transfection. (B) Cells transfected with HA-ERK1 and either vector (Vec) or 22W Raf were replated onto fibronectin (Fn) or maintained in suspension (Sus) for 3 h, as above. Some cells were then treated with 10 ng/ml EGF for 5 min as indicated. HA-ERK was immunoprecipitated (IP) from cell lysates and assayed for activity by in vitro kinase assay using myelin basic protein (MBP) as a substrate. (C) HA-ERK activity was measured in vector and 22W Raf–expressing cells either under nonadherent or adherent conditions at several time points. Values were normalized to the activity of HA-ERK at the 2 h time point on Fn. Shown is the mean and standard deviation of three independent experiments. (D) Cells were cotransfected with equivalent levels of HA-ERK1 and MEK1-ΔED. HA-ERK activity was analyzed, as above, in cells maintained in DMEM/BSA in suspension and adherent conditions for 3 h.

Figure 4

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.

Figure 5

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.

Figure 5

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.

Figure 6

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.

Similar articles

Cited by

References

    1. Adachi M., Fukuda M., Nishida E. Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J. Cell Biol. 2000;148:849–856. - PMC - PubMed
    1. Albanese C., Johnson J., Watanabe G., Eklund N., Vu D., Arnold A., Pestell R.G. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J. Biol. Chem. 1995;270:23589–23597. - PubMed
    1. Aplin A.E., Juliano R.L. Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway. J. Cell Sci. 1999;112:695–706. - PubMed
    1. Aplin A.E., Short S.M., Juliano R.L. Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin alpha chains. J. Biol. Chem. 1999;274:31223–31228. - PubMed
    1. Assoian R.K. Anchorage-dependent cell cycle progression. J. Cell Biol. 1997;136:1–4. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources