Abstract
Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser(34) of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.
Highlights
1-O-alkyl-sn-glycero-3-phosphocholine, the precursor of Platelet-activating factor (PAF), is synthesized from 1-O-alkyl-2-arachidonoylsn-glycero-3-phosphocholine (1-alkyl phosphatidylcholine; PC) by the action of phospholipase A2
Endogenous lyso-PAFAT in inflammatory cells was activated by prophlogistic stimuli [6] and LPCAT2 in mouse peritoneal macrophages was activated by lipopolysaccharide (LPS) stimulation [9]
Because RAW264.7 cells express TLR4 signaling molecules, cells were stimulated with LPS for 30 min, and the lyso-PAFAT activity was examined using the supernatant at 9,000 ϫ g for 10 min
Summary
Materials—PC from frozen egg yolk, LPS from Salmonella minnesota, and anti-FLAG M2 antibody were from Sigma. Preparation of Cell Lysates—Cells were pretreated with or without 20 M MK2 inhibitor III, 20 M SB203580 (p38 MAPK inhibitor), or 1 M (5Z)-7-oxozeaenol (TAK1 (tumor growth factor--activated protein kinase 1) inhibitor) for 1 h and stimulated with 100 ng/ml LPS for 30 min. Cells (peritoneal macrophages or RAW264.7 cells) were washed with ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 0.3 M sucrose, and 1 mM sodium orthovanadate and collected in buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM sodium orthovanadate, 5 mM 2-mercaptoethanol, and 1ϫ EDTA-free Complete. Production of Anti-LPCAT2 and Anti-phospho-LPCAT2 Antibodies—Anti-LPCAT2 antiserum was generated at Immuno-Biological Laboratories (Gunma, Japan). Assay of Lyso-PAF Acetyltransferase and LPCAT—Lyso-PAF acetyltransferase and LPCAT assays were performed as described previously [8, 9]. Sequences of mouse (BAF47695), human (BAF47696), bovine (XP_592529), dog (XP_854080), and rat (XP_001064713) LPCAT2 are available in the DDBJ/EMBL/ GenBankTM databases
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