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.
Highlights
Zhi-Hua Qiu, Marianne S . de Carvalho, and ChristinaC
Phorbol 12-myristate 13-acetate (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, andthe level of phosphorylation increased in response to zymosan, PMA, okadaic acid, and, to a lesser extent, A23187
Summary
Leslie From the Division of Basic Science, Department of Pediatrics, National Jewish Center for Immunology and Respiratory. The 85-kDa PLA2 was phosphorylated on serine in the macrophages, andthe level of phosphorylation increased in response to zymosan, PMA, okadaic acid, and, to a lesser extent, A23187. To determine the effect of dephosphorylation of the PLAz on enzyme activity, the 100,000 X g supernatant fraction (70-90pgof protein) was incubated with 5 pgof acid phosphatasein 100 mM HEPES, pH 6, containing 1 mM dithiothreitol and 2 mM MgCIZat 30"C for 60 min. Phosphorylation of PLAz-Experiments were conducted to determine if the 85-kDa PLA, was phosphorylated in mouse peritoneal macrophages in response to stimuli that induce arachidonic acid release.
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