FIGURE 10.

The S511A/S515A double mutation inhibits stimulated iPLA₂γ activity. COS-1 cells were transiently transfected with M1-GFP-iPLA₂γ WT, GFP-iPLA₂γ S511A/S515A mutant, or empty vector together with COX1. A, after 24 h cells were untreated or were incubated with EGF (100 ng/ml) + ionomycin (Iono, 1.5 μm) for 40 min. PGE₂ release was stimulated significantly by EGF + ionomycin in the cells expressing M1 GFP-iPLA₂γ WT. *, p < 0.0001 versus corresponding untreated group. The increase in PGE₂ release was smaller in cells expressing GFP-iPLA₂γ S511A/S515A mutant. **, p < 0.01 versus cells expressing M1 GFP-iPLA₂γ WT and treated with EGF + ionomycin, four experiments. In these experiments basal PGE₂ release (vector + COX1-transfected, untreated cells) was 191 pg/ml. B, cell lysates were immunoblotted with antibodies to GFP or tubulin. The blot shows comparable levels of expression. C, cell lysates were immunoprecipitated with anti-GFP antibody (+) and were immunoblotted with anti-(R/K)XX(pS/T) or anti-GFP antibodies. The blot shows enhanced phosphorylation of Ser-511 (pS511) in the M1-GFP-iPLA₂γ WT in EGF + ionomycin-stimulated cells. Phosphorylation of Ser-511 is faint in unstimulated WT-expressing cells and is absent in the mutant. D, shown are total lysates of the above immunoprecipitation experiments blotted with anti-GFP antibody.