Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies.
Lipid bodies are inducible lipid domains abundantly present in leukocytes engaged in inflammation. They are rich in esterified arachidonate and are also potential sites for eicosanoid-forming enzyme localization. It is therefore of interest to know whether arachidonate-releasing cytosolic phospholipase A2 (cPLA2) localizes at lipid bodies. Here, we present evidence that cPLA2 and its activating protein kinases, mitogen-activated protein (MAP) kinases, co-localize at lipid bodies. U937 cells express high levels of cPLA2 and contain numerous cytoplasmic lipid bodies. Using double-labeling immunocytochemistry we demonstrated punctate cytoplasmic localizations of both cPLA2 and MAP kinases in U937 cells that were perfectly concordant with fluorescent fatty-acid-labeled lipid bodies. The co-localization of cPLA2 and MAP kinases at lipid bodies was confirmed by subcellular fractionation and immunoblot. Lipid body fractions free of cytosol and other organelles contained significant amounts of [14C]arachidonate-labeled phosphatidylcholine and cPLA2 enzymatic activities. Immunoblotting with specific antibodies identified cPLA2 as well as MAP kinases, including ERK1, ERK2, p85, and p38, in lipid bodies. The co-compartmentalization within arachidonate-rich lipid bodies of cPLA2 and its potentially activating protein kinases suggests that lipid bodies may be structurally distinct intracellular sites active in extracellular ligand-induced arachidonate release and eicosanoid formation.
Yu
W
W
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Bozza
P T
PT
Tzizik
D M
DM
Gray
J P
JP
Cassara
J
J
Dvorak
A M
AM
Weller
P F
PF
D016718:Arachidonic Acid / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002458:Cell Fractionation; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology*; D050356:Lipid Metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure
D002250:Carbon Radioisotopes; D016718:Arachidonic Acid; D019869:Phosphatidylinositol 3-Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2