Abstract
Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.
Highlights
Phospholipases A2 (PLA2)1 catalyze the hydrolysis of the sn-2 ester of membrane phospholipids, the products of which
We investigated how phosphorylation on Ser505 and Ser727 contributes to cytosolic phospholipase A2 (cPLA2) activation, because the phosphorylation on these residues was shown to have the most profound effect on agonist-induced arachidonic acid (AA) release by cPLA2 in mammalian cells [16, 17]
A similar peptide mapping of Ser727-containing peptide revealed that both wild type and S505A had a low level of phosphorylation on Ser727, accurate quantitation was difficult due to the lack of an internal standard. cPLA2 has other potential phosphorylation sites, including Ser437, Ser454, and Ser515
Summary
Materials—1,2-Di-O-hexadecyl-sn-glycerol-3-phosphocholine (DHPC) was purchased from Sigma. 1,2-Dipalmitoyl-sn-glycero-3phosphoglycerol was from Avanti Polar Lipids (Alabaster, AL). 1-Stearoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine ([14C]-SAPC) (55 mCi/mmol) and [3H]AA (200 Ci/mmol) were from Amersham Biosciences and American Radiolabeled Chemicals This equation assumes that each enzyme binds independently to a site on the interface composed of n phospholipids with dissociation constant of Kd. Cell Culture, Transfection, and Protein Production—The vectors for amino-terminal enhanced green fluorescence protein (EGFP) fusion cPLA2 and mutants were generated as described previously [20]. For a transient increase in [Ca2ϩ]i, 150 l of HEK buffer containing 5 M ionomycin and 0.5 mM Ca2ϩ (final concentration) was added to the cells, and cPLA2 translocation was monitored over time. Once cPLA2 and mutants have been translocated to the nuclear envelope (typically ϳ10 min), 100 l of HEK buffer containing 5 mM EGTA (final concentration) was added, and cells were imaged. For searching serine phosphorylation sites, a differential modification of ϩ80 Da on serine was included in the search parameters
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