Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes.
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.
Evans
J H
JH
Program in Cell Biology, Department of Pediatrics, and Department of Immunology, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Spencer
D M
DM
Zweifach
A
A
Leslie
C C
CC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000201:enzymology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D018613:Microscopy, Confocal; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007476:Ionophores; D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D015759:Ionomycin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium