Cloning and expression of cytosolic phospholipase A₂ (cPLA₂) and a naturally occurring variant - Phosphorylation of Ser505 of recombinant cPLA₂ by p42 mitogen-activated protein kinase results in an increase in specific activity

Full-length cytosolic phopholipase A₂ (cPLA₂) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA₂ which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA₂) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA₂ from U937 cells. When cPLA₂ and DcPLA₂ were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA₂ was active in cPLA₂ and lysophospholipase assays while DcPLA₂ was inactive in both assays. cPLA₂ was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [¹²P]Ser(P)505 in cPLA₂ was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA₂. The phosphorylation of cPLA₂ by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.