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Requirement of JNK-Mediated Phosphorylation for Translocation of Group IVA Phospholipase A₂ to Phagosomes in Human Macrophages¹

Javier Casas^*, Clara Meana^*,, Esperanza Esquinas^*,, Martín Valdearcos^*,, José Pindado^*, Jesús Balsinde^2,*, and María A. Balboa^2,*,

^* Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Valladolid, Spain; and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain

Eicosanoids are a broad family of lipids that play a critical role in host defense against bacterial and fungal infections. The first enzyme in the metabolic pathway for the generation of eicosanoids is group IVA phospholipase A₂, also known as cytosolic phospholipase A₂ (cPLA₂). During phagocytosis, cPLA₂ has been found to translocate to the phagosome, although the molecular mechanism involved in such a translocation has not been elucidated. By using enhanced GFP-tagged proteins we show in this work that a nonphosphorylatable cPLA₂ mutant (S505A) does not translocate to the phagosomes, but a mutant that mimics phosphorylation on Ser⁵⁰⁵ (S505E) does it so readily. During phagocytosis, endogenous cPLA₂ is phosphorylated at Ser⁵⁰⁵, and inhibitors of JNK, but not of other related kinases such as p38 or the extracellular-regulated kinases 1 and 2, completely block such a phosphorylation. Inhibition of JNK activity also inhibits the translocation of cPLA₂ to phagosomal membranes, as well as arachidonic acid release to the extracellular medium. Moreover, the S505E mutant makes the enzyme refractory to JNK inhibition, translocating normally to phagosomal membranes. Collectively, these data support a key role for JNK-mediated cPLA₂ phosphorylation at Ser⁵⁰⁵ in the sequence of events leading to translocation and activation of the enzyme to phagosomal membranes in human macrophages.

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.

¹ This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2007-60055 and BFU2007-67154) and by the Regional Government of Castile and León (Grant CSI09-A08).

² Address correspondence and reprint requests to Dr. Jesús Balsinde and Dr. María A. Balboa, Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Calle Sanz y Forés s/n, 47003 Valladolid, Spain. E-mail addresses: jbalsinde{at}ibgm.uva.es and mbalboa{at}ibgm.uva.es

³ Abbreviations used in this paper: cPLA₂, cytosolic phospholipase A₂ (group IVA cytosolic phospholipase A₂); AA, arachidonic acid; bis-BODIPY FL C11-PC, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indecane-3-undecanoyl)-sn-glycero-3-phosphocholine; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced GFP; PIP₂, phosphatidylinositol 4,5-bisphosphate; PLA₂, phospholipase A₂.

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