Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505.
Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 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 cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 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 cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A 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-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.
Abdullah
K
K
Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Merck Frosst Canada Inc., Pointe Claire-Dorval, Quebec.
Cromlish
W A
WA
Payette
P
P
Laliberté
F
F
Huang
Z
Z
Street
I
I
Kennedy
B P
BP
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D010768:Phosphoserine / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017931:DNA Primers; D011994:Recombinant Proteins; D010768:Phosphoserine; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate