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 S podoptera 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 phosphory lated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-7 27 using mass spectrometry and automated Edman sequencing, Sf9 cells i nfected with recombinant virus expressing cPLA(2) exhibited a time-dep endent release of arachidonic acid in response to the calcium ionophor e A23187 or the protein phosphatase inhibitor okadaic acid, which was not observed in Sf9 cells infected with wild-type virus, Stimulation o f Sf9 cells with A23187 and okadaic acid also increased the level of p hosphorylation of cPLA(2). Okadaic acid, but not A23187, induced a gel shift of cPLA(2) and increased the level of phosphorylation of Ser-72 7 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 w hether the same sites on cPLA(2) were phosphorylated in mammalian cell s, 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 digest s of P-32-labeled recombinant cPLA(2) and human monocyte cPLA(2) demon strated that the same peptides on cPLA(2) were phosphorylated in mamma lian cells as in insect cells. These results show that the Sf9-baculov irus expression system is useful for investigation of the phosphorylat ion sites on cPLA(2). The results also suggest that phosphorylation of the cPLA(2) by protein kinases other than mitogen-activated protein k inase may he important for the regulation of arachidonic acid release.