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Regulation of phospholipase A2 activation by phosphorylation in mouse peritoneal macrophages
Z H Qiu1, M S de Carvalho, C C Leslie
1Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
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.
Insights
Phosphorylation of 85-kDa phospholipase A2 (PLA2) activates this enzyme in macrophages, leading to arachidonic acid release. This phosphorylation is crucial for PLA2 activation by various stimuli.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Phospholipase A2 (PLA2) plays a critical role in inflammatory processes by releasing arachidonic acid.
- The precise regulatory mechanisms of PLA2 activation, particularly the role of phosphorylation, remain incompletely understood in macrophages.
Purpose of the Study:
- To investigate the regulation of 85-kDa arachidonoyl-hydrolyzing PLA2 activation by phosphorylation in mouse peritoneal macrophages.
- To elucidate the role of serine phosphorylation in PLA2 activation in response to various stimuli.
Main Methods:
- Stimulation of mouse peritoneal macrophages with phorbol 12-myristate 13-acetate (PMA), okadaic acid, zymosan, and A23187.
- Assessing arachidonic acid release and PLA2 activity in cytosolic fractions.
- Analyzing 85-kDa PLA2 phosphorylation using 2D phosphopeptide mapping and phosphatase treatment.
Main Results:
- PMA and okadaic acid stimulated arachidonic acid release, indicating a role for phosphorylation.
- The 85-kDa PLA2 was phosphorylated on serine, with increased phosphorylation observed upon stimulation with zymosan, PMA, okadaic acid, and A23187.
- PLA2 activity in cytosolic fractions increased in a time-dependent manner following stimulation, and this increase was reversed by phosphatase treatment.
Conclusions:
- Phosphorylation of the 85-kDa PLA2 is a key mechanism for its activation in macrophages.
- Stimuli that induce arachidonic acid release also induce the phosphorylation and activation of 85-kDa PLA2.