Phosphorylation of cytosolic phospholipase A₂ in platelets is mediated by multiple stress‐activated protein kinase pathways

Abstract

Stress-activated protein kinases (SAPKs) are stimulated by cell damaging agents as well as by physiological receptor agonists. In this study we show that human platelets contain the isoforms SAPK2a, SAPK2b, SAPK3 and SAPK4 as determined by immunoblotting with specific antibodies. All four kinases were activated in thrombin-stimulated platelets whereas only SAPK2a and SAPK2b were significantly stimulated by collagen. All four isoforms were able to phosphorylate wild-type human cPLA 2 in vitro, although to different extents, but not cPLA 2 mutants that had Ser505 replaced by alanine. Phosphorylation at Ser505 was confirmed by phosphopeptide mapping using microbore HPLC. SAPK2a and 42-kDa mitogen-activated protein kinase incorporated similar levels of phosphate into cPLA 2 relative to the ability of each kinase to stimulate phosphorylation of myelin basic protein. SAPK2b and SAPK4 incorporated less phosphate, and cPLA 2 was a poor substrate for SAPK3. The inhibitor of SAPK2a and SAPK2b, SB 202190, completely blocked collagen-induced phosphorylation of cPLA 2 at its two phosphorylation sites in vivo, Ser505 and Ser727. We have also reported previously that SB 202190 partially (< 50%) blocks phosphorylation at both sites and to a similar extent in thrombin-stimulated platelets. Inhibition of phosphorylation resulted in a two-to threefold shift to the right in the concentration response curves for arachidonic acid release from thrombin-and collagen-stimulated platelets. Our data suggest that cPLA 2 is a substrate for several SAPK cascades and that phosphorylation of cPLA 2 augments arachidonic acid release.Keywords: arachidonic acid; collagen; cytosolic phospholipase A 2 ; platelet; stress-activated protein kinase; thrombin.The family of mitogen-activated protein kinases (MAPKs) is divided into two major classes, the extracellular-signal regulated protein kinases (ERKs) that are strongly activated by growth factors and tumour-promoting phorbol esters, and the stress-activated protein kinases (SAPKs) that are strongly activated by cellular stresses. The first isoform of the SAPK family, SAPK2a (also known as p38 or p38a) [1], was discovered independently by several groups [2±5]. Strong activators of this kinase include a range of stress stimuli such as hyperosmolarity, heat shock, chemical and oxidative stress and bacterial endotoxins, as well as physiological agents, notably proinflammatory cytokines [1]. Many of its substrates are transcription factors, but SAPK2a also activates a number of kinases, for example MAPK-activated protein kinases-2/3 (MAPKAP-K2/3), which in turn phosphorylates heat shock protein 27 [5,6], the MAPK-interacting kinases-1/2 (Mnk1/2) [7,8], the mitogen-and stress-activated protein kinases (e.g. MSK1) [9] and p38-regulated protein kinases (PRAK) [10]. Several other SAPKs related to SAPK2a have since been discovered. SAPK2b (p38b2) shares approximately 75% amino acid identity with SAPK2a and is similar in its activation profile and substrate specificity [11]. SAPK3 (p38g) [12±14] and SAPK4 (p38...

Serine 727 Phosphorylation and Activation of Cytosolic Phospholipase A2 by MNK1-related Protein Kinases

“…We have recently reported that cPLA2 is phosphorylated to a similar degree on Ser-505 and Ser-727 in human platelets in response to collagen and thrombin and through a pathway that is partially sensitive to inhibitors of p38-2a/2b (16,19). The inhibition data suggest that the Ser-727 kinase is activated by p38-2a/2b in platelets.…”

“…2C, right panel). MNK1 and Related Kinases Phosphorylate cPLA2 on Ser-727 in Vitro-Inhibition of cPLA2 phosphorylation on both Ser-505 and Ser-727 by p38 inhibitors suggests that the Ser-727 kinase may be one of the growing family of protein kinases that are activated by MAPK family members (16,19). cPLA2 in thrombin-and collagen-stimulated platelets is phosphorylated on Ser-505 by one or more p38 isoforms and not by ERK1 or ERK2 P i and then either not stimulated (E) or stimulated (q) with 1 M A23187 for 10 min.…”

“…Retention times in minutes (cpm/peptide standard): oxidized pS727-P (pS727-Pox), 73.2/ 73.8; pS727-P, 79.2/79.1; pS505-P, 102.5/102.8. (17,19). We therefore examined the ability of MNK1, MSK1, PRAK1, and MAPKAP-K2 to phosphorylate cPLA2 in vitro since these kinases are reported to be activated by p38s (24, 25, 36 -38).…”

“…Many investigators have reported that cPLA2 undergoes a shift in electrophoretic mobility following agonist stimulation of cells, an indication of cPLA2 phosphorylation, but phosphopeptide analysis of radiophosphorylated cPLA2-derived peptides has only been carried out with insect cells (Sf9) expressing cPLA2 (20), platelets (16,19), HeLa cells (16) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane for immunoblotting analysis. PRAK1-6His and MSK1-6His (1 g each) were included in gels ii and iii, respectively, as positive controls.…”

“…The degree of inhibition is agonist-dependent, and this appears to be due to the fact that multiple p38s are activated in agonist-specific ways and that only some of these isoforms are sensitive to SB 203580 (19). The inhibition data make it likely that Ser-727 is phosphorylated by a kinase that is downstream of p38s in the activation cascade.…”