Localization of phospholipase C δ3 in the cell and regulation of its activity by phospholipids and calcium

The localization of phospholipase C delta 3 (PLC delta 3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC delta 3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC delta 3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC delta 3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC delta 1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC delta 1 is activated by spermine and sphingosine, whereas PLC delta 3 was inhibited by both these compounds at concentrations that maximally stimulated PLC delta 1. A deletion mutant of PLC delta 3, lacking the entire pleckstrin homology (PH) domain (residues 1-137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC delta 3 activation required calcium ions. The relationship between the Ca2+ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC delta 3 was less sensitive to Ca2+ stimulation. This is in contrast to PLC delta 1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposome assays. We conclude that Ca2+ is necessary to induce specific conformational changes of PLC delta 3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC delta 3 described in this report suggest that PLC delta 3 has a relatively low activity in cellular conditions that fully activate PLC delta 1.