NOVEL FUNCTION OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE AS A COFACTORFOR BRAIN MEMBRANE PHOSPHOLIPASE-D

The activation of phospholipase D (PLD) is a receptor mediated event t hat has been implicated in signal transduction and membrane traffic in eukaryotic cells. Little is known about the biochemical and molecular properties of signal-activated PLDs, and none has been isolated. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) potently stimulates brain membrane PLD activity in vitro in a highly specific m anner. PIP2 increases 10-fold the maximal activity of a partially puri fied PLD with an EC(50) of <0.5 mol %. Other acidic phospholipids, inc luding phosphatidylinositol 4-phosphate, phosphatidylinositol, phospha tidylserine, and phosphatidic acid, are completely or nearly ineffecti ve. Neomycin, a high affinity ligand of PIP2, inhibits membrane-bound PLD but has no effect on the activity of a detergent-solubilized or pa rtially purified enzyme. The addition of PIP2 restores the sensitivity of partially purified PLD to neomycin inhibition, indicating that neo mycin blocks membrane PLD activity by binding to endogenous PIP2, Thes e results define a novel function of PIP2 as a cofactor for brain memb rane PLD and suggest that PLP(2) synthesis and hydrolysis could be imp ortant determinants in regulating PLD action in signal transduction an d membrane transport.