title: Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes.
abstract: The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A2 (cPLA2) were identified using recombinant cPLA2 expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of tryptic digests of 32P-labeled recombinant cPLA2 showed four major peaks of radiolabeled phosphopeptides. The phosphorylated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-727 using mass spectrometry and automated Edman sequencing. Sf9 cells infected with recombinant virus expressing cPLA2 exhibited a time-dependent release of arachidonic acid in response to the calcium ionophore A23187 or the protein phosphatase inhibitor okadaic acid, which was not observed in Sf9 cells infected with wild-type virus. Stimulation of Sf9 cells with A23187 and okadaic acid also increased the level of phosphorylation of cPLA2. Okadaic acid, but not A23187, induced a gel shift of cPLA2 and increased the level of phosphorylation of Ser-727 by 4.5-fold, whereas the level of phosphorylation of the other sites increased by 60% or less in response to both agonists. To determine whether the same sites on cPLA2 were phosphorylated in mammalian cells, human monocytes were studied. Okadaic acid stimulation of monocytes induced a gel shift of cPLA2, increased the release of arachidonic acid, and increased the level of phosphorylation of cPLA2 on serine residues. Comparison of two-dimensional peptide maps of tryptic digests of 32P-labeled recombinant cPLA2 and human monocyte cPLA2 demonstrated that the same peptides on cPLA2 were phosphorylated in mammalian cells as in insect cells. These results show that the Sf9-baculovirus expression system is useful for investigation of the phosphorylation sites on cPLA2. The results also suggest that phosphorylation of the cPLA2 by protein kinases other than mitogen-activated protein kinase may be important for the regulation of arachidonic acid release.
authors.affiliation: Division of Basic Science, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: de Carvalho
authors.forename: A L
authors.initials: AL
authors.lastname: McCormack
authors.forename: E
authors.initials: E
authors.lastname: Olson
authors.forename: F
authors.initials: F
authors.lastname: Ghomashchi
authors.forename: M H
authors.initials: MH
authors.lastname: Gelb
authors.forename: J R
authors.initials: JR
authors.lastname: Yates
authors.forename: C C
authors.initials: CC
authors.lastname: Leslie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010449:Peptide Mapping; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D016718:Arachidonic Acid; D011494:Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin