Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2alpha in regulating interfacial kinetics and binding and cellular function

Abstract

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA(2)alpha (IMLF(-/-)) were reconstituted with wild type and cPLA(2)alpha mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA(2)alpha translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA(2)alpha to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA(2)alpha to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF(-/-) expressing phosphorylation site (S505A) and PIP(2) binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA(2)alpha, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA(2)alpha reduced activation by PIP(2) but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA(2)alpha hydrolytic activity.

FIGURE 1.

Effect of PMA and serum on [Ca²⁺]_i and AA release in IMLF^+/+. A, [³H]AA-labeled IMLF^+/+ were stimulated with 10% serum (right) or 100 nm PMA (left) and [³H]AA release was determined at given times and compared with unstimulated (US) cells. [³H]AA released into the medium was measured and expressed as a percentage of the total cellular radioactivity in each well. The results are the average of three experiments ± S.E. for PMA (left) and two experiments ± S.D. for serum (right). B, live cell calcium imaging of IMLF^+/+ loaded with the fluorescent calcium indicator FuraRed-AM was measured after the addition (arrows) of serum, PMA, or vehicle (DMSO). Calcium changes are shown in individual cells stimulated with 10% serum without (panel 1) or with (panel 2) a 15-min preincubation in EGTA. Panel 3 shows calcium oscillations in cells stimulated with 100 nm PMA for 30 min. Panel 4 illustrates calcium changes in cells stimulated with PMA with an EGTA preincubation. Panel 5 shows control cells (incubated without EGTA) using DMSO vehicle in place of PMA. Calcium changes were determined by correcting for background fluorescence values from each cell and calculated as a ratio of bound to unbound calcium fluorescence intensities (F403/F470). Data are presented relative to time 0 (R_T/R₀), and starting (R_T/R₀) for each cell is set at 1. Each line represents data from an individual cell. Graphs are representative of a minimum of three independent experiments.

FIGURE 2.

ERK and p38 activation by serum and PMA in IMLF^+/+. Cell lysates of unstimulated (US) IMLF^+/+ or cells stimulated with 10% serum or 100 nm PMA were prepared at given times after stimulation. Activation of ERKs or p38 (A) or phosphorylation of cPLA₂α on Ser-505 (B) was determined by Western blotting using phosphospecific antibodies. Sample loading was determined using total ERK antibodies (data not shown) or antibodies to total cPLA₂α (B). Results are representative of three independent experiments. C, Western blots of lysates of serum-stimulated IMLF^-/- expressing either wild type ECFP-cPLA₂α (WT) or EYFP-cPLA₂αS505A were probed with antibodies to total cPLA₂α or phosphospecific antibodies to cPLA₂α phosphorylated on Ser-505. The Western blot confirms the specificity of the phosphospecific antibodies for cPLA₂α phosphorylated on Ser-505.

FIGURE 3.

MAPK activation is required for AA release in IMLF^+/+ in response to serum and PMA. [³H]AA-labeled IMLF^+/+ were preincubated with 10 μm SB203580 (15 min), 10 μm U0126 (15 min), 1 μm wortmannin (30 min), 10 μm KN93 (30 min), 10 μm GF109203X (60 min), or 15 ng/μl CHX (30 min) or no treatment followed by stimulation with serum for 10 min (A, C, and E) or PMA for 45 min (B, D, and F). [³H]AA released into the medium was measured and presented as a percentage of total cellular [³H]AA. G, IMLF^+/+ were preincubated with SB203580, U0126, or CHX or no treatment followed by stimulation with serum for 10 min or PMA for 45 min, as described above. Cell lysates of unstimulated (US) or stimulated IMLF^+/+ were analyzed by Western blotting to determine activation of ERKs (p-ERK) or phosphorylation of cPLA₂α on Ser-505 (p-cPLA₂α) using phosphospecific antibodies. Sample loading was determined using antibodies to total cPLA₂α or antibodies to β-tubulin. Data represent the average of three experiments ± S.E. (A-D), representative of three independent experiments in duplicate ± S.D. (E and F) or a representative Western blot of three independent experiments (G).

FIGURE 4.

Reconstitution of [³H]AA release by IMLF^-/- in response to serum and PMA by expression of cPLA₂ α. IMLF^-/- were incubated for 26 h with different amounts of adenoviruses for expression of EGFP-cPLA₂α (A) and ECFP-cPLA₂α (B). The adenovirus stock for expression of EGFP-cPLA₂α has a lower titer than the virus stock for ECFP-cPLA₂α and therefore required higher volumes to achieve similar levels of expression. Cells labeled with [³H]AA were washed and then left unstimulated (US) or treated with serum for 10 min (A) or with PMA for 45 min (B). [³H]AA released into the medium was determined and presented as a percentage of the total cellular radioactivity. Western blots of lysates collected from each well show the corresponding levels of cPLA₂α expression. Results are representative of three independent experiments conducted in duplicate.

FIGURE 5.

Calcium is required for AA release but not ERK and p38 activation in response to serum and PMA. A, IMLF^+/+ were preincubated with 10 mm EGTA for 15 min and left unstimulated (US) or treated with serum for 10 min or PMA for 45 min. [³H]AA released into the medium was measured and presented as a percentage of total cellular radioactivity. B, IMLF^+/+ were preincubated with or without EGTA and stimulated with serum or PMA for the indicated times. Immunoblots were conducted using equal protein per lane and probed with phosphospecific antibodies against phospho-ERK and phospho-p38 or with antibody to total ERKs to determine sample loading. C, [³H]AA-labeled IMLF^-/- expressing either wild type ECFP-cPLA₂α, EYFP-cPLA₂αD43N or neither (uninfected) were stimulated with 10% serum (10 min) or 100 nm PMA (45 min), and [³H]AA release was compared with unstimulated cells. Wells with matching expression levels of cPLA₂α wild type and mutant (inset) determined by Western blot analysis were used for [³H]AA release determination. Results are representative of a minimum of three independent experiments conducted in duplicate. The release of [³H]AA by the mutant was significantly less (p < 0.05) than by wild type cPLA₂α (WT), as indicated (^*).

FIGURE 6.

Serum-stimulated cPLA₂α translocation to Golgi correlates with [Ca²⁺]_i increase and is dependent on a functional C2 domain. A, IMLF^-/- expressing EGFP-cPLA₂α were incubated in phenol red free DMEM and stimulated with 10% serum. Live cell images were collected every 3 s using an FITC filter and a ×40 oil immersion objective. Images are representative of 10 individual experiments. B, IMLF^-/- expressing EGFP-cPLA₂α were incubated in phenol red-free DMEM, fixed 2 min after stimulation with serum, and then probed with anti-giantin primary antibody and Texas Red secondary antibody to visualize Golgi. C, IMLF^-/- expressing EGFP-cPLA₂α were loaded with FuraRed-AM, and live cell images were collected using FITC, F403, and F470 filters after stimulation with serum (arrow). D, IMLF^-/- expressing EGFP-cPLA₂α were incubated in medium containing EGTA, and then images were collected using a FITC filter after stimulation with serum (arrow). E, translocation data from C and D were analyzed to determine the percentage of EGFP-cPLA₂α bound to Golgi at the peak of serum-induced translocation (∼30 s) in cells incubated with and without extracellular EGTA. Translocation data were calculated based on average fluorescence intensity of EGFP-cPLA₂α on the Golgi in each cell. Values were corrected for background fluorescence and differential bleaching at each wavelength through the duration of the imaging and expressed relative to time 0 (F_T/F₀). Calcium ratios (F403/F470) were calculated and corrected for background fluorescence and expressed relative to time 0 (R_T/R₀). Graphs are representative of 10 cells from three independent experiments. F, IMLF^-/- co-expressing ECFP-cPLA₂α and EYFP-cPLA₂αD43N were stimulated with serum, and translocation was determined as described in A. Data are presented relative to time 0 (F_T/F₀). The graph is representative of 15 cells from three independent experiments.

FIGURE 7.

PMA stimulates ECFP-cPLA₂α translocation to Golgi. A, IMLF^-/- expressing ECFP-cPLA₂α were incubated in phenol red-free DMEM and stimulated with PMA. Live cell images were collected every 30 s using a CFP filter and a ×40 oil immersion objective. Images are representative of 10 individual cells. B, IMLF^-/- expressing EGFP-cPLA₂α were incubated in phenol red-free DMEM, fixed 15 min after stimulation with 100 nm PMA, and then probed with anti-giantin primary antibody and Texas Red secondary antibody to visualize Golgi. C, IMLF^-/- co-expressing wild type ECFP-cPLA₂α and EYFP-cPLA₂αD43N were stimulated with PMA (arrow), and images were collected using both a CFP and YFP filter. Translocation data were calculated based on average fluorescence intensity of a mask of the Golgi in each cell. Values were calculated by subtracting background fluorescence and correcting for differential bleaching at each wavelength through the duration of the imaging. Data are presented relative to time 0 (F_T/F₀). Results are representative of five independent experiments and 10 individual cells.

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.

FIGURE 9.

A, binding of cPLA₂α-PAP and cPLA₂αS505P to PAPC vesicles. Binding solutions contained 1 ml of buffer A, 0.5 mg/ml BSA, and 200 μm PAPC as vesicles and 50 ng of cPLA₂α-PAP (filled circles) or cPLA₂αS505P (open circles). After ultracentrifugation, two 100-ml aliquots of the supernatant were each submitted to the standard radiometric cPLA₂α assay to determine the amount of enzyme not bound to vesicles. The latter is plotted as the percentage of enzyme added to each binding solution, where 100% corresponds to the radiometric assay signal measured for 5 ng of cPLA₂α added directly from the stock solution to the radiometric assay mixture (see “Experimental Procedures”). Each experimental condition was carried out in duplicate, and both data points are plotted. B, hydrolysis of [¹⁴C]PAPC vesicles by cPLA₂α-PAP, cPLA₂αS505P, and cPLA₂αS505P/S727P versus the concentration of calcium. Reactions contained 0.1 ml of buffer A, 0.5 mg/ml BSA, and 200 μm [¹⁴C]PAPC as vesicles and 200 ng of cPLA₂α-PAP (filled circles, solid line) or cPLA₂αS505P (open circles, solid line) or cPLA₂αS505P/S727P (open squares, dashed line). Reactions were quenched after 2 min at 37 °C. Each experimental condition was carried out in duplicate, and both data points are plotted.

FIGURE 10.

Role of basic residues in cPLA₂α catalytic domain in regulating translocation and AA release in response to serum and PMA. [³H]AA-labeled IMLF^-/- expressing either wild type ECFP-cPLA₂α or EYFP-cPLA₂αK488N/K543N/K544N were stimulated for 10 min with serum (A) or 45 min with PMA (B), and [³H]AA release was measured and expressed as a percentage of the total cellular radioactivity in each well. The release of [³H]AA by the mutant was significantly less (p < 0.05) than by wild type cPLA₂α, as indicated (^*). Cell lysates were made from each well, and immunoblotting for cPLA₂α was conducted to determine expression levels (inset) of wild type and mutant cPLA₂α in each well. C, IMLF^-/- co-expressing wild type ECFP-cPLA₂α and EYFP-cPLA₂αK488N/K543N/K544N were stimulated with serum, and images were collected using both a CFP and YFP filter and a ×40 oil immersion objective. Translocation data were calculated based on average fluorescence intensity of a mask of the Golgi in each cell. Values were calculated by subtracting background fluorescence and correcting for differential bleaching at each wavelength throughout the imaging. Data are presented relative to time 0 (F_T/F₀). Data are representative of 10 individual cells from three independent experiments.