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doi: 10.1242/10.1242/jcs.00146

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Nuclear localisation of cytosolic phospholipase A₂- in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation

¹ School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
² Cancer Research UK Clinical Centre, St. James' Hospital, University of Leeds, Leeds, LS9 7TF, UK

View larger version (58K): [in a new window]   Fig. 1. Distribution of cPLA- in confluent and subconfluent EA.hy.926 cells. (A) cPLA- was detected by western blotting EA.hy.926 lysates (20 µg protein) using an affinity-purified goat polyclonal antibody (lane 1). Also shown are control lanes corresponding to antigen-adsorbed antibody (lane 2) and HRP anti-goat IgG controls (lane 3). (B) Cells (1x10 for subconfluent and 5x10 for confluent) were seeded onto glass coverslips, and cPLA- was detected by immunofluorescence microscopy. Bar, 10 µm. (C) Densitometrical plot analysing the distribution of staining across individual cells (marked by dashed line in B). (D) Peptide-adsorbed antibody control. (E) Detection of NuMA in confluent and subconfluent cells.

View larger version (30K): [in a new window]   Fig. 2. cPLA2- in nuclear fractions. (A) Nuclei from subconfluent and confluent cells were isolated and analysed by immunofluorescence microscopy. Also shown are nuclei obtained from A23187-stimulated cells (5 µM, 10 minutes). (B) 20 µg of total homogenate and nuclear samples were western blotted, and LDH and cPLA- were detected using goat polyclonal antibodies. (C) Quantification of the amounts of cPLA- in nuclear fractions expressed as a percentage of the amount in the total sample±s.e.m. (n=3).

View larger version (37K): [in a new window]   Fig. 3. Effects of serum starvation on cPLA- localisation. (A) cPLA- was detected by immunofluorescence microscopy in cells starved for 0, 24 and 48 hours. Bar, 10 µm. (B) Graph showing the changes in intensity of nuclear staining in sections taken through the nuclei of cells following serum starvation (in arbitrary units, n=90, data represents averages from three independent experiments±s.e.m.). (C) Cells starved for 24 and 48 hours were re-fed with complete medium, grown for a further 24 hours and viewed by immunofluorescence microscopy. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 4. Effects of staurosporine treatment on the nuclear localisation of cPLA-. Cells (1x10 for sub-confluent, 5x10 for confluent samples) were seeded onto coverslips and grown overnight. Cells were then treated with 1 µM staurosporine in HEPES/Tyrode's buffer for 30 minutes at 37°C (control cells were incubated with buffer alone). cPLA- was detected by immunofluorescence microscopy. Bar, 10 µm. (B) The intensity of cPLA- staining in sections through the nuclei of confluent (i) and subconfluent (ii) cells was quantified. Plots represent average intensities (in arbitrary units)±s.e.m. (n=90, data from three independent experiments).

View larger version (66K): [in a new window]   Fig. 5. Effects of okadaic acid pre-treatment on the levels of nuclear cPLA-. (A) Cells (5x10) were seeded onto coverslips and grown to confluency. Cells were then washed with PBS and pre-treated with 1 µM okadaic acid in HEPES/Tyrode's buffer for 30 minutes at 37°C (controls cells were incubated with buffer alone). Cells were then fixed and permeabilised and cPLA- was detected by immunofluorescence microscopy. Bar, 10 µm. (B) Plot showing the change in the level of nuclear intensity (±s.e.m., n=90, data from three independent experiments) in sections taken through control and okadaic acid treated cells.

View larger version (28K): [in a new window]   Fig. 6. Effects of the specific MEK and p38MAPK inhibitors, PD98059 and SB203580, on the levels of nuclear cPLA-. (A) Cells (1x105) were seeded onto coverslips and grown overnight. Cells were then pre-treated with PD98059 or SB203580 (20 µM in HEPES/Tyrode's buffer) for 30 minutes at 37°C (control cells were incubated with buffer alone). cPLA- was detected by immunofluorescence microscopy. Bar, 10 µm. (B) The level of nuclear staining in sections taken through the control, PD98059- and SB203580-treated cells were quantified. The plot shows average intensity (in arbitrary units)±s.e.m. (n=90, data from three independent experiments). (C) Nuclei were isolated from control and PD98059-, SB203580- and staurosporine-treated cells. 20 µg of total homogenate (T) and nuclear (N) proteins were analysed by western blotting. (D) Quantification of the relative amounts of cPLA2- present in the nuclear and total fractions (expressed as a percentage of the total±s.e.m., n=3)

View larger version (36K): [in a new window]   Fig. 7. Effects of the edible mushroom Agaricus bisporus lectin (ABL) on the serum-induced nuclear import of cPLA-. Cells were grown on coverslips and starved for 24 or 48 hours. Cells were then re-fed with complete medium or pre-treated with ABL (20 µg/ml) for 6 hours prior to the addition of complete medium. (A) cPLA- was detected by immunofluorescence microscopy. Bar, 10 µm. (B) Graph showing changes in intensity of nuclear staining (in arbitrary units, n=90, data represents averages from three independent experiments±s.e.m.) in sections taken through serum-starved, re-fed and ABL-treated re-fed cells.

View larger version (36K): [in a new window]   Fig. 8. Effects of leptomycin B (LMB) on the nuclear export of cPLA-. Cells were grown overnight on coverslips then serum starved for 24 hours. The effects of LMB were examined by treating the cells for 1 hour with 10 ng/ml LMB prior to serum starving. (A) cPLA- was detected in control cells (a), serum-starved cells (b) and LMB-treated serum-starved cells (c). Bar, 10 µm. (B) Graph showing changes in intensity of nuclear staining (in arbitrary units, n=90, data represents averages from three independent experiments±s.e.m.) in sections taken through the nuclei of cells.

View larger version (78K): [in a new window]   Fig. 9. Putative cPLA2- nuclear localisation and export signals. (A) Amino-acid sequence of human cPLA2-. The potential NLSs are highlighted in blue and green. The potential nuclear export signal is outlined in yellow. (B) 3D structure of cPLA2-, showing the positions of the potential NLSs and NES in their respective colours. Also highlighted are the approximate locations of the phosphorylation sites, Ser505 and Ser727.