Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505

Abstract Cytosolic PLA 2 (cPLA 2 ) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA 2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA 2 , the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA 2 overexpressed in Sf9 cells. The level of overexpressed CPLA 2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA 2 , Ser-505 was replaced by Ala, and this mutant (CPLA 2S505A ) was expressed in Sf9 cells. Analysis of the overexpressed CPLA 2S505A showed that it migrated only as the lower unshifted cPLA 2 band confirming that the baculovirus overexpressed cPLA 2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA 2 with phorbol 12-tetradecanoate 13-acetate (TTA) shifted all of the overexpressed cPLA 2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [ 32 P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA 2 and CPLA 2s5o5A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl- sn -glycero-3-phospho-choline as substrate. Under these conditions the specific activity of cPLA 2 was, 2 μmol/min per mg, whereas cPLA 2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA 2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpessed cPLA 2 is a very good source for the Ser-505 phosphorylated enzyme.