Phospholipase C-δ3 binds with high specificity to phosphatidylinositol 4,5-bisphosphate and phosphatidic acid in bilayer membranes

Abstract

In order to acquire an understanding of phospholipase C-δ3 (PLC-δ3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC-δ3 to large, unilamellar phospholipid vesicles (LUVs). PLC-δ3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC-δ3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 x 105 M-1. High binding of PLC-δ3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC-δ3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC-δ3 to PA and PtdSer vesicles was 1.6 X 104 M-1 and 9.4 X 102 M-1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC-δ3 to the phospholipid membrane. Binding of PLC-δ3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration-dependent increase in activity of PLC-δ3. The stimulatory effect of PA on PLC-δ3 was calcium-dependent. At Ca2+ concentrations lower than 1 μM, no effect of PA on the activity of PLC-δ3 was observed. PA enhanced PLC-δ3 activity by increasing the V(max) and lowering K(m) for PtdInsP2. As the mol fraction of PA increased from 0-40 mol% the enzyme V(max) increased 2.3-fold and K(m) decreased threefold. Based on the results presented, we assume that PA supports binding of PLC-δ3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium-dependent interaction with the C2 domain of PLC-δ3. We propose that binding of PLC-δ3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC-δ3 activity.