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J. Biol. Chem., Vol. 278, Issue 42, 41431-41442, October 17, 2003

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Mechanism of Group IVA Cytosolic Phospholipase A₂ Activation by Phosphorylation^*

Sudipto Das , John D. Rafter , Kwang Pyo Kim , Steven P. Gygi  and Wonhwa Cho  ¶

From the Department of Chemistry, University of Illinois, Chicago, Illinois 60607 and the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

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 µM 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 µM 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.

Received for publication, May 9, 2003 , and in revised form, July 25, 2003.

^* This work was supported by National Institutes of Health Grants GM52598 and GM53987. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho{at}uic.edu.

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