title: Cloning and expression of cystolic 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.
abstract: Full-length cytosolic phospholipase 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 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. [32P]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.
authors.affiliation: Zeneca Pharmaceuticals, Macclesfield, UK.
authors.forename: R D
authors.initials: RD
authors.lastname: Gordon
authors.forename: I A
authors.initials: IA
authors.lastname: Leighton
authors.forename: D G
authors.initials: DG
authors.lastname: Campbell
authors.forename: P
authors.initials: P
authors.lastname: Cohen
authors.forename: A
authors.initials: A
authors.lastname: Creaney
authors.forename: D C
authors.initials: DC
authors.lastname: Wilton
authors.forename: D J
authors.initials: DJ
authors.lastname: Masters
authors.forename: G A
authors.initials: GA
authors.lastname: Ritchie
authors.forename: R
authors.initials: R
authors.lastname: Mott
authors.forename: I W
authors.initials: IW
authors.lastname: Taylor
authors.forename: K R
authors.initials: KR
authors.lastname: Bundell
authors.forename: L
authors.initials: L
authors.lastname: Douglas
authors.forename: J
authors.initials: J
authors.lastname: Morten
authors.forename: M
authors.initials: M
authors.lastname: Needham
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D007313:Insecta / Q000166:cytology; D007963:Leukocytes, Mononuclear / Q000201:enzymology / Q000502:physiology; D008223:Lymphoma / Q000201:enzymology / Q000473:pathology; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology; D019319:Okadaic Acid; D010741:Phospholipases A / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D054467:Phospholipases A2; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D006005:Phosphorylases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014020:Tissue Extracts; D014407:Tumor Cells, Cultured
chemical_list: D004988:Ethers, Cyclic; D011994:Recombinant Proteins; D014020:Tissue Extracts; D019319:Okadaic Acid; D006005:Phosphorylases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D010741:Phospholipases A; D054467:Phospholipases A2; D010749:Phosphoprotein Phosphatases