Phosphatidylinositol 4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells

Abstract

The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A2 (also known as cytosolic phospholipase A2alpha, cPLA2alpha). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA2alpha translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca2+. However, cPLA2alpha also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca2+. How cPLA2alpha interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes translocation of cPLA2alpha to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca2+ concentration. PtdIns(4,5)P2 anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.

Figure 1.

EGFP-cPLA₂α translocation and AA release driven by TR-PIP₂. EGFP-cPLA₂-(A, B, and E) or EGFP (C)-transfected HEK cells were treated with TR-PI(4,5)P₂/histone (A and C), TR-PI(3,4)P₂/histone (E), or carrier only (histone; B). Pictures were taken at the indicated time points under the confocal microscope. (D) [³H]AA-labeled HEK cells were either untreated (open circles) or treated with TR-PI(4,5)P₂/carrier (closed circles), and [³H]AA release was measured at the indicated time periods. Each experiment was performed at least three times with identical results.

Figure 2.

TR-PIP₂ effects on RAW 264.7 cells. RAW 264.7 macrophages transfected with EGFP-cPLA₂ were treated with TR-PI(4,5)P₂/histone for the indicated periods of time and analyzed by confocal microscopy (A) or for [³H]AA release (B) as in Figure 1. Each experiment was performed at least three times with identical results.

Figure 3.

Ca²⁺ is necessary for EGFP-cPLA₂α translocation in response to TR-PI(4,5)P₂. EGFP-cPLA₂-transfected HEK cells were treated with TR-PI(4,5)P₂/histone in HBSS medium without Ca²⁺, and pictures were taken under the confocal microscope. After a 30-min treatment, 1.3 mM Ca₂Cl was added to the incubation medium (the two columns on the right-hand side), and pictures were taken at the indicated time points. Each experiment was performed at least three times with identical results.

Figure 4.

A intact Ca²⁺ binding domain is necessary for EGFP-cPLA₂ translocation in response to TR-PI(4,5)P₂. HEK cells transfected with the mutant EGFP-D43N-cPLA₂ were treated with TR-PI(4,5)P₂/histone in HBSS with 1.3 mM CaCl₂ and pictures were taken at the indicated time points by confocal microscopy. Each experiment was performed at least three times with identical results.

Figure 5.

EGFP-cPLA₂ and TR-PI(4,5)P₂ colocalization analysis. HEK cells transfected with the construct EGFP-cPLA₂ were treated with TR-PI(4,5)P₂ for 20 min in the presence (A) or absence (D) of extracellular calcium, and pictures of green and red fluorescences were taken by confocal microscopy. The merge of both fluorescences is also shown. LaserPix software analysis of colocalization fluorescences in A is shown as a white mask (B) or as a colored three-dimensional representation, where red means maximum level of colocalization (255) and dark blue means the minimum (0) (C). Each experiment was performed at least three times with identical results.

Figure 6.

Intracellular Ca²⁺ levels in HEK cells. Intracellular measurements of Ca²⁺ were performed in Fluo-3-loaded HEK cells treated with TR-PI(4,5)P₂/histone (A) or 5 μM ionomycin (B) in buffer containing 1.3 mM CaCl₂. Arrows indicate the time point at which the stimuli were added. Each data point represents the average of 20–40 cells. Fluorescence of Fluo-3 was detected by microscopy and recorded every 15–30 s. Each experiment was performed at least three times with identical results.

Figure 7.

Intracellular Ca²⁺ requirements for TR-PI(4,5)P₂-driven translocation of EGFP-cPLA₂. Fluo-3-loaded HEK cells were first treated with TR-PI(4,5)P₂/histone (A) or 5 μM ionomycin (B) in a buffer without Ca²⁺ and later, different CaCl₂ amounts were added as indicated. Fluorescence of Fluo-3 was detected by microscopy and recorded every 15–30 s. Each data point represents the average of 20–40 cells. Translocation of EGFP-cPLA₂ was analyzed by confocal microscopy under the same conditions indicated above for HEK cells [C, cells treated with TR-PI(4,5)P₂/histone; D, cells treated with 5 μM ionomycin]. Extracellular concentration of CaCl₂ is indicated. Each experiment was performed at least three times with identical results.

Figure 8.

Analysis of the EGFP-4KE/A-cPLA₂α mutant. (A) HEK cells transfected with the mutant EGFP-4KE/A-cPLA₂α were treated with TR-PI(4,5)P₂/histone, and pictures were taken under the confocal microscope for the indicated time periods. (B) Colocalization analyses (LaserPix software) of the fluorescence from EGFP4KE/A-cPLA₂α mutant and TR-PI(4,5)P₂ or EGFP-cPLA₂α and TR-PI(4,5)P₂ as a control, were performed in the presence of absence of extracellular calcium, as indicated. Colocalization index of red to green (black bars) or green to red (gray bars) is shown. Each data bar represents the average of 10–20 cells. Experiments were performed at least three times with identical results.

Figure 9.

AA released by HEK cells transfected with the mutant EGFP-4KE/A-cPLA₂α. [³H]AA-labeled HEK cells transfected with the EGFP-cPLA₂α construct (closed circles) or the mutant EGFP-4KE/A-cPLA₂α (open circles) were treated with TR-PI(4,5)P₂/carrier and [³H]AA release was measured at the time indicated time points. Each experiment was performed at least three times with identical results.