Cloning and expression of cytosolic phospholipase A(2) (cPLA(2)) and a naturally occurring variant - Phosphorylation of Ser505 of recombinant cPLA(2) by p42 mitogen-activated protein kinase results in an increase in specific activity

Full-length cytosolic phopholipase A(2) (cPLA(2)) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA(2) which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA(2)) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA(2), from U937 cells. When cPLA, and DcPLA(2) 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 cPLA(2) was active in cPLA, and lysophospholipase assays while DcPLA(2) was inactive in both assays. cPLA(2) 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, [P-32]Ser(P)505 in cPLA(2) 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 cPLA(2). The phosphorylation of cPLA(2) by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.