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 delta 1 (PLC delta 1), The rate of PLC delta 1 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 PLC delta 1 from 5.4 to 0.5 mu M. In the presence of Ca2+, PLC delta 1 specifically bound to PS/PC but nob to PA/PC vesicles in a dose-dependent and saturable manner. Ca2+ also bound to PLC delta 1 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 mu M. 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 PLC delta 1 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.