Regulation of cytosolic phospholipase A2 by phosphorylation.

Introduction Phospholipases catalyse the cleavage of membrane phospholipids and thereby generate second messengers that participate in intracellular signal transduction processes or act as precursors of tissue hormones. Cytosolic phospholipase Az (cPLA2) is an 85 kDa enzyme that cleaves arachidonic acid at the sn-2 position of the phospholipid [1,2]. The intracellular location of the enzyme enables it to mediate receptor-regulated release of arachidonic acid [3]. Arachidonic acid is the precursor for prostaglandins, thromboxanes and leukotrienes, and lysophospholipid can be metabolized to yield platelet-activating factor. These substances exhibit diverse physiological, tissue-specific activities by participating in haemostatic and inflammatory responses and in the regulation of vascular tone, blood pressure, uterine and glandular function. The activity of cPLA2 is regulated by an increase in the intracellular Ca2+ concentration and by phosphorylation. Ca2+ mediates binding of the enzyme to phospholipid structures without being involved in the catalytic mechanism itself. An increase in the intracellular Ca2+ concentration triggers translocation of cPLA2 to cellular membranes through a Ca2+-dependent lipidbinding motif [2,4]. cPLA2 contains a consensus sequence for mitogen-activated protein kinase (MAPK), Pro-Leu-Ser-505-Pro. Ser-505 is phosphorylated by ~ 4 2 " ' " ~ ~ in vitro and in cells coexpressing cPLA2 and p42mapk, as determined by the comparison of wild-type cPLA2 with mutated Ser-505 +Ala-505 cPLAz [ S ] . Phosphorylation by ~ 4 2 ' " " ~ ~ increases the intrinsic activity of the lipase by 2to 3-fold as measured in vitro using phosphatidylcholine vesicles or sonicated liposomes as substrate [5,6]. Concomitant activation of ~42/p44" ' "~~ and cPLA2 is observed in many cells. cPLA2 is also phosphorylated in vitro by protein kinase C and by protein kinase A. However, depending on the conditions of the lipase assay, phosphorylation by protein kinase C has little if any effect on cPLAz activity; phosphoryla-