Modulation of the activity of cytosolic phospholipase A 2 ( cPLA 2 ) by cellular sphingolipids and inhibition of cPLA 2 by sphingomyelin

This article is available online at http://www.jlr.org Arachidonic acid (AA) is a precursor of eicosanoids, including prostaglandins, thromboxanes, and leukotrienes, playing an important role in several physiological functions ( 1 ). The biosynthesis of these AA metabolites occurs mainly through the activation of phospholipase A2 (PLA2) in response to a wide variety of stimuli such as cytokines, growth factors, and neurotransmitters ( 2 ). PLA2 catalyzes hydrolysis of the sn-2 position of glycerophospholipids to release free AA. Mammalian cells have structurally diverse forms of PLA2 including secretory PLA2, Ca 2+ -independent PLA2, and cytosolic PLA2 (cPLA2) ( 3, 4 ). Among these PLA2s, the 85 kDa cPLA2, specifi cally cPLA2 , is highly selective for glycerophospholipids containing AA. cPLA2 is regulated mainly by an increase in the intracellular Ca 2+ concentrations ([Ca 2+ ]i) and by the phosphorylation on serine residues by mitogen-activated protein kinase (MAPK) ( 3, 4 ). The binding of Ca 2+ to the C2 domain of cPLA2 triggers translocation of cPLA2 from the cytosol to the perinuclear region including the Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope. cPLA2 can be phosphorylated at Ser 505 , Ser 515 , and Ser 727 , which increases its intrinsic enzymatic activity 2to 3-fold in vitro ( 5–8 ). Abstract We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2 (cPLA2 ) using two Chinese hamster ovary (CHO)-K1-derived mutants defi cient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca 2+ ionophore A23187 increased 2-fold and 1.7fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2 inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2 . Phosphatidylcholine vesicles containing SM reduced cPLA2 activity. Furthermore, SM disturbed the binding of cPLA2 to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2 -dependent release of AA by inhibiting the binding of cPLA2 to glycerophospholipids. —Nakamura, H., S. Wakita, A. Suganami, Y. Tamura, K. Hanada, and T. Murayama. Modulation of the activity of cytosolic phospholipase A2 (cPLA2 ) by cellular sphingolipids and inhibition of cPLA2 by sphingomyelin. J. Lipid Res . 2010. 51: 720–728.