Activation of phospholipase C δ1 through C2 domain by a Ca²⁺-enzyme- phosphatidylserine ternary complex

The concentration of free Ca²⁺ and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C δ1 (PLCδ1). The rate of PLCδ1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated-20-fold-by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca²⁺ concentration required for half-maximal activation of PLCδ1 from 5.4 to 0.5μM. In the presence of Ca²⁺, PLCδ1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca²⁺ also bound to PLCδ1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca²⁺ concentration required for half-maximal Ca²⁺ binding was estimated to be 8 μM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646654 from the C2 domain of PLCδ1 impaired Ca²⁺ binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca²⁺-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.