title: Regulation of phospholipase A2 activation by phosphorylation in mouse peritoneal macrophages.
abstract: The regulation of phospholipase A2 (PLA2) activation by phosphorylation, and phosphorylation of an 85-kDa, arachidonoyl-hydrolyzing PLA2 was investigated in mouse peritoneal macrophages. Phorbol 12-myristate 13-acetate (PMA) and okadaic acid, an inhibitor of serine/threonine phosphatases, stimulated arachidonic acid release, supporting a role for phosphorylation events in regulating PLA2 activation. In response to zymosan, PMA, or A23187, arachidonic acid was released at a linear rate up to 30-45 min after stimulation, whereas there was a 30-min lag preceding arachidonic acid release in response to okadaic acid. The 85-kDa PLA2 was phosphorylated on serine in the macrophages, and the level of phosphorylation increased in response to zymosan, PMA, okadaic acid, and, to a lesser extent, A23187. Two-dimensional phosphopeptide mapping revealed multiple phosphopeptides, several of which showed increased phosphorylation in response to zymosan, okadaic acid, and PMA. Zymosan, PMA, A23187, or okadaic acid stimulated time-dependent increases in PLA2 activity in the cytosolic fraction. PLA2 activation was most rapid in response to PMA, whereas activation in response to okadaic acid was delayed similar to the time course of arachidonic acid release. The cytosolic PLA2 had characteristics of the 85-kDa enzyme, including kinetic properties and substrate preference. Phosphatase treatment of the cytosols dephosphorylated the 85-kDa PLA2 and reversed the increase in activity. The results provide evidence that phosphorylation of the 85-kDa PLA2, induced by stimuli that induce arachidonic acid release, is an important mechanism for activation of the enzyme in macrophages.
authors.affiliation: Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
authors.forename: Z H
authors.initials: ZH
authors.lastname: Qiu
authors.forename: M S
authors.initials: MS
authors.lastname: de Carvalho
authors.forename: C C
authors.initials: CC
authors.lastname: Leslie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation; D004988:Ethers, Cyclic / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D010449:Peptide Mapping; D010529:Peritoneal Cavity / Q000166:cytology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
chemical_list: D004988:Ethers, Cyclic; D019319:Okadaic Acid; D016718:Arachidonic Acid; D000001:Calcimycin; D015054:Zymosan; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate