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 membrane traffic. We have previously shown that phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) stimulates in vitro partially purified brain membrane PLD activity, defining a novel function of PtdIns-4,5-P2 as a PLD cofactor. In the present study we extend these observations to permeabilized U937 cells. In these cells, the activation of PLD by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) is greatly potentiated 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-P2 synthesis by phosphoinositide kinases. As expected, MgATP was absolutely required for maintaining elevated phosphatidylinositol 4-phosphate (PtdIns-4-P) and PtdIns-4,5-P2 levels in the permeabilized cells. In the presence of MgATP, GTP gamma S further elevated the levels of the phosphoinositides. The importance of PtdIns-4,5-P2 for PLD activation was examined by utilizing a specific inhibitory antibody directed against phosphatidylinositol 4-kinase (PtdIns 4-kinase), the enzyme responsible for the first step in the synthesis of PtdIns-4,5-P2. Anti-PtdIns 4-kinase completely inhibited PtdIns 4-kinase activity in vitro and reduced by 75-80% PtdIns-4-P and PtdIns-4,5-P2 levels in the permeabilized cells. In parallel, the anti-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-P2 levels are required for PLD activation. This conclusion is supported by the fact that neomycin, a high affinity ligand of PtdIns-4,5-P2, also blocked PLD activation. Furthermore, the activity of PLD in U937 cell lysate was stimulated by PtdIns-4,5-P2 in a dose-dependent manner. The current results indicate that PtdIns-4,5-P2 synthesis is required for PLD activation in permeabilized U937 cells and strongly support the proposed function of PtdIns-4,5-P2 as a cofactor for PLD. In addition, the results further establish PtdIns-4,5-P2 as a key component in the generation of second messengers via multiple pathways including phosphoinositide-phospholipase C, phosphoinositide 3-kinase and PLD.
Pertile
P
P
Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts.
Liscovitch
M
M
Chalifa
V
V
Cantley
L C
LC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D009355:Neomycin / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis* / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D000255:Adenosine Triphosphate; D010739:Phospholipase D; D009355:Neomycin