Abstract
Increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) promote cytosolic phospholipase A(2) (cPLA(2)) translocation to intracellular membranes. The specific membranes to which cPLA(2) translocates and the [Ca(2+)](i) signals required were investigated. Plasmids of EGFP fused to full-length cPLA(2) (EGFP-FL) or to the cPLA(2) C2 domain (EGFP-C2) were used in Ca(2+)/EGFP imaging experiments of cells treated with [Ca(2+)](i)-mobilizing agonists. EGFP-FL and -C2 translocated to Golgi in response to sustained [Ca(2+)](i) greater than approximately 100-125 nm and to Golgi, ER, and perinuclear membranes (PNM) at [Ca(2+)](i) greater than approximately 210-280 nm. In response to short duration [Ca(2+)](i) transients, EGFP-C2 translocated to Golgi, ER, and PNM, but EGFP-FL translocation was restricted to Golgi. However, EGFP-FL translocated to Golgi, ER, and PNM in response to long duration transients. In response to declining [Ca(2+)](i), EGFP-C2 readily dissociated from Golgi, but EGFP-FL dissociation was delayed. Agonist-induced arachidonic acid release was proportional to the [Ca(2+)](i) and to the extent of cPLA(2) translocation. In summary, we find that the differential translocation of cPLA(2) to Golgi or to ER and PNM is a function of [Ca(2+)](i) amplitude and duration. These results suggest that the cPLA(2) C2 domain regulates differential, Ca(2+)-dependent membrane targeting and that the catalytic domain regulates both the rate of translocation and enzyme residence.
Highlights
The Ca2ϩ-sensitive cytosolic phospholipase A21 is found in most cells and tissues and hydrolyzes phospholipids containing arachidonate at the sn-2 position to liberate arachidonic acid (AA), an important regulator of diverse cell functions and a precursor of potent inflammatory lipids [1]
Cytosolic PLA2 Translocates to the Golgi, Endoplasmic Reticulum, and a Perinuclear Membrane—Previous immunocytochemical and GFP fusion protein studies have consistently described the pattern of cytosolic phospholipase A2 (cPLA2) translocation in response to a saturating [Ca2ϩ]i as perinuclear [10, 11, 13,14,15,16,17], but the specific membranes in the perinuclear region targeted by cPLA2 have not been definitively identified
Previous studies have shown that EGFP fused to the amino terminus of Calcium Signals Regulating cPLA2 Translocation
Summary
EGFP-cPLA2 Fusion Constructions—DNA encoding human cPLA2 was cloned into the vector pCR2.1 as previously described [11]. Cell Culture—MDCK cells obtained from ATCC were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 0.292 mg/ml glutamine (growth medium) in 5% CO2 at 37 °C. Dual Imaging Digital Microscopy of EGFP Translocation and [Ca2ϩ]i Changes—Stably transfected cells grown on MatTek plates were washed with HHBSS containing 1 mM probenecid and incubated with 10 M Fura2-AM (Calbiochem) in HHBSS, 1 mM probenecid, and 1% Me2SO for 1.5 h at 37 °C. Single-cell imaging was performed on a Nikon inverted microscope using a 40ϫ, 1.3 NA oil immersion objective, Fura and FITC filter sets (Chroma), and a CCD camera (Cooke). 2, 3, J–L, and 4, D–G and K–N) were performed using an inverted Nikon microscope using a 60ϫ, 1.4 NA oil immersion objective, a Photometrics CCD camera, FITC filters (Chroma), and IP Labs software. Cells were scraped in 0.5 ml of 0.1% Triton X-100 for determining the total cellular radioactivity
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