FIGURE 8.

AA release and translocation of cPLA₂α phosphorylation site mutants. Parallel cultures of [³H]AA-labeled IMLF^-/- expressing either wild type ECFP-cPLA₂α (WT) or EYFP-cPLA₂α phosphorylation site mutants as indicated were stimulated for 10 min with serum (A and B) or for 45 min with PMA (E and F). [³H]AA released into the medium is expressed as a percentage of the total cellular radioactivity in each well. Immunoblotting was conducted to determine expression levels of wild type and mutant cPLA₂α in each well (insets). [³H]AA release is shown from wells with matching expression levels. The release of [³H]AA by the mutants was significantly less (p < 0.05) than by wild type cPLA₂α, as indicated (^*). Live cell images of IMLF^-/- co-expressing wild type ECFP-cPLA₂α and either EYFP-cPLA₂αS505A/S727A (C) or EYFP-cPLA₂αS505A (D) were collected every 3 s after serum stimulation, using CFP and YFP filters and a ×40 oil immersion objective. Translocation to Golgi in cells expressing wild type (gray lines) or mutant cPLA₂α (black lines) is shown for two representative cells. Values are corrected for background fluorescence and differential bleaching and are presented relative to time zero (F_T/F₀). Data are representative of 20 individual cells from three independent experiments.