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

Robert D. Gordon, Ian A. Leighton, David G. Campbell, Philip Cohen, Ann Creaney, David C. Wilton, David J. Masters, Graham A.F. Ritchie, Richard Mott, Ian W.F. Taylor, Kenneth R. Bundell, Lynne Douglas, John Morten, Maurice Needham (Lead / Corresponding author)

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22 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)690-697
Number of pages8
JournalEuropean Journal of Biochemistry
Volume238
Issue number3
DOIs
Publication statusPublished - 1 Jun 1996

Keywords

  • cytosolic phospholipase A
  • kinase
  • lysophospholipase
  • phosphatase
  • phosphorylation

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