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First published online 29 March 2005
doi: 10.1242/jcs.02308

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Activation of either ERK1/2 or ERK5 MAP kinase pathways can lead to disruption of the actin cytoskeleton

Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK

View larger version (95K): [in a new window]   Fig. 1. Activation of ERK5 leads to disruption of the actin cytoskeleton. (A) MEK5DD activates ERK5 but not ERK1/2. Expression vectors for HA-tagged ERK5 and Flag-tagged ERK2 were co-transfected with expression plasmids for HA-MEK5DD, Myc-H-RasV12 or empty vector. After 24 hours cells were lysed and analysed by SDS-PAGE followed by western blotting for epitope tags, ERK5 or phospho-ERK. The two top arrows indicate the slower migrating phosphorylated form of ERK5 (phosphorylated) and non-phosphorylated ERK5. (B,C) NIH3T3 cells were microinjected, in the presence of serum, with expression vectors for activated MEK1 (MEK1EE) and ERK2, activated MEK5 (MEK5DD) and ERK5. 3 hours post-injection, cells were starved of serum and after 24 hours, plates were fixed and stained for polymerised actin with Texas Red-phalloidin and for the expressing proteins with antibodies against MEK1, ERK2, MEK5 and ERK5. Arrows indicate microinjected cells. Bar, 20 µm.

View larger version (92K): [in a new window]   Fig. 2. ERK5 and ERK1/2 act differently to disrupt the actin cytoskeleton. Activation of ERK5 does not block the ability of constitutively activated RhoA to stimulate the formation of actin stress fibres. (A) NIH3T3 cells were microinjected, in the presence of serum, with expression vectors for constitutively activated RhoA (RhoAV14) alone or in combination with activated MEK1 (MEK1EE) + ERK2 or activated MEK5 (MEK5DD) + ERK5. 3 hours post-injection cells were serum starved and after 24 hours were fixed and stained with ERK2 and ERK5 antibodies. RhoAV14 expression was recognised with either a mouse or rabbit RhoA antibody and polymerised actin was detected using Texas Red-phalloidin. Arrows indicate injected cells; (B) treatment with 1 µM PD184352 restores the actin cytoskeleton in Ras-transformed NIH3T3 cells (clone 149169). 149169 cells were treated for 24 hours with 1 µM PD184352 or vehicle, permeabilised, fixed and stained for polymerised actin as in A and for vinculin, as a marker of focal adhesions, with a mouse monoclonal antibody followed by a anti-mouse FITC-coupled antibody. Bar, 20 µm.

View larger version (79K): [in a new window]   Fig. 3. Src activates the ERK5 pathway. (A) Activated Src (Y527F) was transfected together with different combinations of wild-type and mutant ERK5 and/or MEK5, the MEF2D luciferase reporter and a ß-galactosidase plasmid for normalisation. Results are the mean±s.e. of eight independent experiments. The decrease in luciferase activation in the Y527F Src + ERK5AEF sample was statistically significant (*) by the Student's t-test (P=0.01). Arrows indicate nuclei of microinjected cells. (B) Activated Src causes translocation of ERK5 from the cytoplasm to the nucleus. NIH3T3 cells were microinjected at 5:1 ratio with empty vector and an EGFP expression plasmid (upper panels) or activated Y527F Src and EGFP (lower panels). Cells were then stained with ERK5 antibody to detect endogenous ERK5. Src protein expression was inferred by GFP fluorescence. Arrows indicate nuclei of microinjected cells. (C) NIH3T3 cells were microinjected with HA-MEK5DD and stained with anti-HA and anti-ERK5 to detect endogenous ERK5. (D) ERK5 is present in the nucleus of Src-transformed cell. Different v-Src transformed NIH3T3 clones were serum starved for 24 hours and then stained as in B. Bar, 20 µm.

View larger version (17K): [in a new window]   Fig. 4. Activation of ERK5 is required for transformation by oncogenic Src. (A) NIH3T3 cells were transfected with either Y527F Src alone, or with varying amounts of expression plasmids for MEK5AA, wild-type ERK5 or ERK5AEF. After 11-16 days transformed foci were blind scored. (B) NIH3T3 cells were transfected with either of two variants of oncogenic HER2/Neu (neu* or P1*) (see Materials and Methods) alone or with varying amounts of MEK5AA plasmid. Appearance of transformed foci was scored as in A.

View larger version (58K): [in a new window]   Fig. 5. Inhibition of both the ERK1/2 and ERK5 pathways is required to restore the cytoskeleton in Src-transformed cells. (A) Src-transformed NIH3T3 cells (D4F9-ac2M) were treated for 24 hours with PD184352 or solvent alone then fixed and stained with Texas Red-phalloidin to visualise filamentous actin and a vinculin antibody to visualise focal adhesions. (B) Lysates of Src-transformed NIH3T3 cells treated for up to 48 hours with PD184352 (1=1 µM and 10=10 µM PD184352) or solvent alone (D=DMSO) were western blotted with antibodies against activated dually phosphorylated ERK1/2 and ß-tubulin and pan-ERK as loading controls. (C) v-Src transformed NIH3T3 cells (D4F9-ac2M) were microinjected with expression vectors for HA-MEK5AA, Flag-ERK5AEF or MEK1A. After 24 hours, cells were fixed and stained for HA-MEK5AA and Flag-ERK5AEF with HA (12CA5) and Flag (M2) directed antibodies. MEK1A expression was detected with a MEK1 antibody. Polymerised actin was stained with Texas Red-phalloidin. Bar, 20 µm.

View larger version (17K): [in a new window]   Fig. 6. Inhibition of ERK1/2 signalling enhances ERK5 activation by Src. NIH3T3 cells were transfected with expression vectors for Y527F Src, dominant-negative MEK1 (MEK1A), dominant-negative MEK5 (MEK5AA) or ERK5 (ERK5AEF), where indicated, cells were treated with 1 µM PD184352 (PD1) or 10 µM (PD10) for 24 hours.