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First published online 16 March 2004
doi: 10.1242/jcs.01040

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MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation

Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (71K): [in a new window]   Fig. 1. ERK5 is localized in the nucleus in Rat-1 cells. (A) Rat-1 cells were grown on coverslips, fixed with PFA, permeabilized with 0.2% Triton X-100 and stained with rabbit anti-ERK5 antibody directed to the C terminus of ERK5 in the presence or absence of blocking peptide (50 µg/ml). (B) Rat-1 cells were treated as described above and stained with antibodies to different epitopes of ERK5 (N-19, C-20, H-300).

View larger version (59K): [in a new window]   Fig. 2. ERK5 is localized in the nucleus before and after stimulation. HeLa cells (A) or Rat-1 cells (B) were grown on coverslips, serum-starved and activated with 50 ng/ml EGF for the indicated times. The treated cells were then fixed and stained with anti-ERK5 antibody (N-19). Then the cells were tested for ERK5 phosphorylation. For this purpose, HeLa cells (C) or Rat-1 cells (D) were grown in 6 cm tissue culture plates and activated with 10 ng/ml EGF for the indicated times. After treatment, the cells were harvested with RIPA buffer, and cell lysates were separated by SDS-PAGE and subjected to western blots analysis with anti-ERK5 or anti-phospho-ERK5 (P-ERK5) antibodies. Biochemical fractionation of ERK5 was performed as described under Materials and Methods. (E) HeLa cells (upper panels) or Rat-1 cells (lower panels), were grown in 10 cm tissue culture plates, serum-starved and activated with EGF (50 ng/ml, 15 minutes). Then the cells were washed with ice cold PBS and fractionated into nuclear and cytoplasmic fractions. Equal volume aliquots of each fraction were subjected to SDS-PAGE and western blotting and analyzed with anti-ERK5 (C terminus) and anti-phospho-ERK5 (P-ERK5) antibodies. (F) The samples described in E were subjected to western blot analysis with anti-Sp-1 and anti-caspase 3 antibodies.

View larger version (51K): [in a new window]   Fig. 3. Exogenous GFP-ERK5 is localized in the cytoplasm. (A) HeLa cells were grown on coverslips, transfected with GFP-ERK5 construct (2.5 µg) or GFP-ERK5 (2.5 µg) together with either HA-WT-MEK5, HA-AA-MEK5 or HA-MEK5D (2.5 µg each) using the PEI method (Materials and Methods) and left untreated for additional 24 hours. This was followed by serum-starvation (0.1%) for 18 hours, after which the cells were left either unstimulated (NS) or stimulated with EGF (50 ng/ml, 30 minutes). Then, the cells were washed with PBS, fixed with PFA, and the GFP was visualized using a fluorescence microscope. (B) HeLa cells were grown in 6 cm tissue culture plates and transfected with GFP-ERK5 alone (4 µg) and together with either WT-MEK5 (WT, 4 µg) or HA-MEK5D (D, 4 µg). As above, the cells were left to recover, serum-starved, and either stimulated with EGF (50 ng/ml, 30 minutes) or left unstimulated (NS). Then the cells were harvested and subjected to western blotting with anti P-ERK5 and GFP antibodies.

View larger version (78K): [in a new window]   Fig. 4. Endogenous ERK5 is retained in the nucleus after in situ detergent extraction. (A) HeLa cells were grown on coverslips as described before, serum-starved and stimulated with EGF (50 ng/ml, 15 minutes) or left untreated. The cells were then subjected to an in situ detergent extraction with NP-40 (Materials and Methods, 0.2%, 5 minutes). The fixed cells were stained with goat anti-ERK5 antibody (N19) and with DAPI. Phase contrast micrographs of the non-activated NP-40 untreated and treated cells demonstrates the effect of the detergent extraction. Staining with anti-histone H1 antibodies is shown as a positive control in the lower panel. (B) Rat-1 cells were grown and treated as described in A, except that the in situ NP-40 extraction used 0.5% for 10 minutes.

View larger version (35K): [in a new window]   Fig. 5. GFP-ERK5 accumulates in the nucleus without anchoring upon LMB treatment. (A) HeLa cells were grown on coverslips and transfected with GFP-ERK5 construct (2.5 µg DNA) using the PEI method. The cells were either treated with LMB (5 ng/ml, 1 hour) or left untreated (NT), and then were washed with PBS, fixed with 3% PFA, permeabilized with 0.2% Triton X-100 and stained with DAPI. (B) HeLa cells were grown and treated as in A. After LMB treatment, cells were subjected to an in situ detergent extraction with NP-40 (0.2%, 5 minutes), stained with DAPI and examined with a fluorescence microscope.

View larger version (26K): [in a new window]   Fig. 6. MEK5 is localized in the nucleus. (A) HeLa cells were grown as described above, serum-starved, and then either stimulated with EGF (50 ng/ml, 15 minutes) or left unstimulated (NS). Then the cells were washed, fixed and stained with anti-MEK5 antibody and DAPI. (B) Rat-1 cells were grown and stimulated as described above, and then stained with anti-MEK5 antibody and DAPI. A blocking peptide (50 µg/ml) was added to the staining (lower panel) for competition. (C) Specificity of the anti-MEK5 antibody was demonstrated by western blot with extracts (30 µg) from HeLa and Rat-1 cells.

View larger version (48K): [in a new window]   Fig. 7. MEK5 is retained in the nucleus of resting but not of stimulated cells. HeLa (A) or Rat-1 (B) cells were grown on coverslips as described above, serum-starved and either stimulated with EGF (50 ng/ml, 15 minutes) or left unstimulated (NS). Then the cells were subjected to an in situ extraction with NP-40 (0.2% for HeLa and 0.5% for Rat-1, 5 minutes), after which the cells were fixed and stained with anti-MEK5 antibody and DAPI.

View larger version (23K): [in a new window]   Fig. 8. LMB does not affect ERK5 activation but does affect its down regulation. HeLa cells were grown in 6 cm plates, serum-starved and preincubated with LMB (5 ng/ml, 1 hour) or left untreated (NT), followed by EGF stimulation (50 ng/ml) for the indicated times. Then, the cells were harvested in RIPA buffer and the lysates were separated by SDS-PAGE and subjected to a western blot analysis with anti-ERK5 antibody (N-19).

View larger version (63K): [in a new window]   Fig. 9. MEKK2 translocates to the nucleus upon EGF stimulation. A. HeLa and Rat-1 cells were grown on coverslips, serum-starved and either stimulated with EGF (50 ng/ml) for the indicated times or left unstimulated (NS). Then the cells were washed, fixed and stained with anti-MEKK2 antibody (N-19) and with DAPI. (B) HeLa and Rat-1 cells were grown as for A, and incubated with LMB (5 ng/ml, 1 hour) followed by stimulation (EGF, 50 ng/ml) for the indicated times or left without FGF stimulation. Then the cells were washed, fixed and stained with anti-MEKK2 antibody and with DAPI.