Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A(2) expressed in insect cells and present in human monocytes

The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A(2) (cPLA(2)) were identified using recombinant cPLA, expressed in Spodoptera frugiperda (Sf9) cells, Analysis by high performance liquid chromatography of tryptic digests of P-32-labeled recombinant cPLA(2) 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 cPLA(2) 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 cPLA(2). Okadaic acid, but not A23187, induced a gel shift of cPLA(2) 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 cPLA(2) were phosphorylated in mammalian cells, human monocytes were studied, Okadaic acid stimulation of monocytes induced a gel shift of cPLA(2), increased the release of arachidonic acid, and increased the level of phosphorylation of cPLA(2) on serine residues, Comparison of two-dimensional peptide maps of tryptic digests of P-32-labeled recombinant cPLA(2) and human monocyte cPLA(2) demonstrated that the same peptides on cPLA(2) 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 cPLA(2). The results also suggest that phosphorylation of the cPLA(2) by protein kinases other than mitogen-activated protein kinase may he important for the regulation of arachidonic acid release.