PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE SYNTHESIS IS REQUIRED FOR ACTIVATION OF PHOSPHOLIPASE-D IN U937 CELLS

Phospholipase D (PLD) has been implicated in signal transduction and m embrane traffic. We have previously shown that phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P-2) stimulates in vitro partially purified brain membrane PLD activity, defining a novel function of PtdIns-4,5- P-2 as a PLD cofactor. In the present study we extend these observatio ns to permeabilized U937 cells. In these cells, the activation of PLD by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) is greatly potent iated by MgATP. We have utilized this experimental system to test the hypothesis that MgATP potentiates PLD activation by G proteins because it is required for PtdIns-4,5-P-2 synthesis by phosphoinositide kinas es. As expected, MgATP was absolutely required for maintaining elevate d phosphatidylinositol 4-phosphate (PtdIns-4-P) and PtdIns-4,5-P-2 lev els in the permeabilized cells. In the presence of MgATP, GTP gamma S further elevated the levels of the phosphoinositides. The importance o f PtdIns-4,5-P-2 for PLD activation was examined by utilizing a specif ic inhibitory antibody directed against phosphatidylinositol 4 kinase (PtdIns 4-kinase), the enzyme responsible for the first step in the sy nthesis of PtdIns-4,5-P-2. Anti-PtdIns 4-kinase completely inhibited P tdIns 4-kinase activity in vitro and reduced by 75-80% PtdIns-4-P and PtdIns-4,5-P-2 levels in the permeabilized cells. In parallel, the ant i-PtdIns 4-kinase fully inhibited the activation of PLD by GTP gamma S and caused a 60% inhibition of PLD activation by the phorbol ester 12 -O-tetradecanoylphorbol-13-acetate, indicating that elevated PtdIns-4, 5-P-2 levels are required for PLD activation. This conclusion is suppo rted by the fact that neomycin, a high affinity ligand of PtdIns-4,5-P -2, also blocked PLD activation. Furthermore, the activity of PLD in U 937 cell lysate was stimulated by PtdIns-4,5-P-2 in a dose-dependent m anner. The current results indicate that PtdIns-4,5-P-2 synthesis is r equired for PLD activation in permeabilized U937 cells and strongly su pport the proposed function of PtdIns-4,5-P-2 as a cofactor for PLD. I n addition, the results further establish PtdIns-4,5-P-2 as a key comp onent in the generation of second messengers via multiple pathways inc luding phosphoinositide-phospholipase C, phosphoinositide 3-kinase and PLD.