Group IV cytosolic phospholipase A2 binds with high affinity and specificity to phosphatidylinositol 4,5-bisphosphate resulting in dramatic increases in activity

The group TV cytosolic phospholipase A(2) (cPLA(2)) exhibits a potent and specific increase in affinity for lipid surfaces containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) at physiologically relevant concentrations. Specifically, the presence of 1 mol % PtdIns(4,5)P-2 in phosphatidylcholine vesicles results in a 20-fold increase in the binding affinity of cPLA(2). This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P-2 binds to cPLA(2) in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P-2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P-2 on the activity of cPLA(2) is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA(2) with PtdIns(4,5)P-2 and the interaction of the pleckstrin homology domain of phospholipase C delta(1) with PtdIns(4,5)(2) combined with sequence analysis of cPLA(2) lead us to propose the existence and location of a pleckstrin homology domain in cPLA(2). We further show that the very nature of the interaction of proteins such as cPLA(2) with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data.