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First published online March 2, 2007
Journal of Experimental Biology 210, 1036-1045 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02719

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Activation and nuclear translocation of ERK in response to ligand-dependent and -independent stimuli in liver and gill cells from rainbow trout

Hannes L. Ebner, Michael Blatzer, Muhammad Nawaz and Gerhard Krumschnabel^*

Institut für Zoologie und Limnologie, and Center of Molecular Biosciences, Leopold Franzens Universität Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria

View larger version (74K): [in this window] [in a new window]   Fig. 1. Amino acid alignment of trout tMEK, deduced from the determined complete coding region of the nucleotide sequence, and carp cMEK1 isoform (GenBank accession no. AB063390), human hMAP2K2 (BC018645), rat rMAP2K2 (NM_205388), and carp cMKK4 (AB063389). Specific protein interaction sites indicated are: putative MAPK docking site (amino acids 1–27; underlined), NES-sequence (AA 36–47, boxed), and catalytic domain (AA 70–364, grey background); amino acid numbering refers to trout MEK.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Dynamics of ERK phosphorylation in primary hepatocytes (A), RTgill-W1 cells (B) and RTH-149 hepatoma cells (C) from rainbow trout exposed to hypo-osmolarity, 30 nmol l–1 EGF or 10 µmol l–1 CuCl2, for up to 60 min as indicated. Numbers below the blots are changes in pERK abundance, expressed relative to controls at time zero, denoted as means ± s.e.m. of at least 3 independent experiments. *Statistically significant difference from the control value, as assessed on non-normalized data (P<0.05). c60', controls incubated in standard conditions for 60 min. Activated ERK was determined by western blot analysis using an antibody against the dually phosphorylated form of ERK. The main band detected with this antibody migrated at approximately 44 kDa. In A, the second panel shows an example of a western blot analysis of total ERK abundance using an antibody against the phosphorylated and non-phosphorylated form of ERK (tERK). Similar analyses were made for all cell types under all conditions and indicated that ERK abundance was not altered by the treatments over the time course studied.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Cellular distribution of phosphorylated ERK in primary hepatocytes (A), RTgill-W1 cells (B) and RTH-149 hepatoma cells (C) from rainbow trout stimulated by exposure to hypo-osmolarity (Hypo), EGF or Cu, in the absence or presence of leptomycin B (LB), as indicated. Cells were stained with an antibody against the dually phosphorylated form of ERK and a FITC-labelled secondary antibody and were then examined by confocal laser scanning microscopy. The right panel in the top row in A shows a negative control (Neg.) stained in the absence of the primary antibody. Scale bars, 20 µm (valid for all images in B and C).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Quantitative estimation of nuclear translocation of phosphorylated ERK in RTgill-W1 cells, as determined from the ratio of staining intensity measured in the nucleus to that of a cytoplasmic region of the cells. (A) Time course of the change in nuclear/cytoplasmic ratio following the addition of EGF is shown in controls and in cells after pre-incubation with leptomycin B. Values are means ± s.e.m. of at least 20 cells from 2–3 independent cultures. (B) Nuclear/cytoplasmic fluorescence ratio of cells after exposure to hypo-osmotic shock, EGF or Cu at the time where the maximum increase of relative nuclear fluorescence was determined (5' or 15'). Values are means ± s.e.m. of at least 16 cells from 2–3 independent cultures. Asterisks indicate a statistical significant difference (P<0.05) from the value at time zero (A) or from cells in the absence of LB (B).

View larger version (25K): [in this window] [in a new window]   Fig. 5. (A) Dynamics of MEK phosphorylation, determined by western blotting, in RTgill-W1 cells exposed to 30 nmol l–1 EGF for up to 60 min. Figures below the blot indicate changes in pMEK abundance, expressed relative to controls at time zero, and denote means ± s.e.m. of four independent experiments. c60, controls incubated in standard conditions for 60 min. (B) Cellular distribution of pMEK in RTgill-W1 cells incubated in standard conditions or exposed to EGF for 5 min. The right image shows cells pre-incubated with leptomycin B (LB) for 60 min prior to addition of EGF. Cells were stained with an antibody against the dually phosphorylated form of MEK and a FITC-labelled secondary antibody and were then examined by confocal laser scanning microscopy. Scale bar, 20 µm (valid for all three images). (C) Quantitative estimation of nuclear/cytoplasmic distribution of phosphorylated MEK, expressed as nuclear/cytoplasmic ratio, in RTgill-W1 cells exposed to EGF with or without prior pre-incubation with LB. Values are means ± s.e.m. of at least 14 cells from three independent cultures. For both treatments, nuclear/cytoplasmic ratio is significantly decreased at all time points compared to the value at time zero (P<0.05).