Identification of the Phosphorylation Sites of Cytosolic Phospholipase A2 in Agonist-stimulated Human Platelets and HeLa Cells

Abstract

The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 Pphosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA 2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-␣ or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stressactivated protein kinase (p38 mapk ), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38 mapk inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA 2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA 2 expressed in a baculovirus/insect cell system to cPLA 2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA 2 in different mammalian cells stimulated with multiple agonists.Cytosolic phospholipase A 2 (cPLA 2 ) 1 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids (1, 2). The 85-kDa enzyme is present in many mammalian cells (3), and strong evidence is accumulating for the role of cPLA 2 in the generation of tissue mediators that are metabolites of arachidonic acid, such as prostaglandins, leukotrienes, and thromboxanes. In contrast to the small molecular weight phospholipase A 2 s that are secreted and are active on the outside of cells, cPLA 2 is regulated by intracellular signals that are propagated from surface receptors. One important regulatory mechanism appears to be a rise in the intracellular Ca 2ϩ concentration which causes translocation of cPLA 2 from the cytosol to internal membranes (4 -7) where it binds through a Ca 2ϩ -dependent lipid-binding domain (8). A second, well established mechanism of the regulation of cPLA 2 activity is by phosphorylation on Ser-505 through a mitogen-activated protein kinase (MAPK) (9) which modestly increases the intrinsic activity of the lipase measured in vitro (3, 10, 11). Phosphorylation of cPLA 2 together with release of arachidonic acid has been observed in a variety of cells (11)(12)(13)(14)(15)(16).A thorough characterization of the phosphorylation sites of human cPLA 2 heterologously expressed in Spodoptera frugiperda (Sf9) cells by high performance liquid chromatography (HPLC), mass spectrometry, and protein sequencing has revealed four sites of phosphorylation: Ser-...

A Requirement for Calcium-independent Phospholipase A2 in Thrombin-induced Arachidonic Acid Release and Growth in Vascular Smooth Muscle Cells

“…Earlier studies by other investigators have shown that p38 MAPK plays a role in thrombin-induced cPLA 2 phosphorylation and activation in platelets (35). Recently, we have reported that p38 MAPK via phosphorylation and activation of ATF-1 is involved in thrombin-induced growth in VSMC (36).…”

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets

“…Previous studies that have studied cPLA 2 activation in thrombin stimulated platelets have commonly used concentrations of thrombin at 1 to 2 IU/ml [3,24,28]. We constructed a concentrationresponse curve to thrombin and observed significant increases in PLA 2 activity at concentrations of thrombin greater than 0.005 IU/ml (data not shown).…”

“…Two synergistic pathways for cPLA 2 activation have been implicated; increased cytosolic Ca 2+ results in translocation of cPLA 2 to the membrane, and p38 MAP kinase phosphorylates the serine residues on the enzyme [2,3,24]. Activation of both pathways is required for arachidonic acid liberation from membrane phospholipids in response to thrombin stimulation.…”

“…Additionally, a cytosolic PLA 2 (cPLA 2 ) that requires micromolar concentrations of intracellular Ca 2+ for translocation to membrane phospholipids has been demonstrated in human platelets [2]. In response to thrombin stimulation, platelet cPLA 2 is phosphorylated and activated by p38 MAP kinase [3]. To date, there is little evidence for the involvement of a Ca 2+ -independent PLA 2 (iPLA 2 ) in human platelets.…”

Functional Interaction of Calcium-/Calmodulin-dependent Protein Kinase II and Cytosolic Phospholipase A2

“…p42/p44 mitogen-activated protein (MAP) kinase phosphorylate cPLA 2 on Ser-505 in many cell types (6,20), whereas other members of the MAP kinase family, namely p38 stress-activated protein kinases, phosphorylate cPLA 2 in platelets (21). Rigorous studies to map the phosphorylation sites on cPLA 2 have been carried out only with platelets, HeLa cells, and CHO cells, and these studies reveal that cPLA 2 is phosphorylated on Ser-505 and Ser-727 (22). More recent work has shown that Ser-727 phosphorylation is catalyzed by MNK1 or a closely related isoform, which is a protein kinase that is activated by members of the MAP kinase family, and that both Ser-505 and Ser-727 phosphorylation are required for full activation of cPLA 2 (23).…”