LOCALIZATION OF PHOSPHOLIPASE-C DELTA-3 IN THE CELL AND REGULATION OFITS 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 si gnificantly lower than that in the membrane fraction. In rat liver, PL C delta 3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on P LC delta 3 revealed that this enzyme is inhibited by phosphatidylethan olamine (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 Ptd Cho and PtdEtn. In a detergent assay, PLC delta 1 is activated by sper mine and sphingosine, whereas PLC delta 3 was inhibited by both these compounds at concentrations that maximally stimulated PLC delta 1. A d eletion 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 th e same extent as the native enzyme. PLC delta 3 activation required ca lcium ions. The relationship between the Ca2+ concentration and enzyma tic activity was almost identical for the deletion mutant and the nati ve enzyme. However, in the liposome assay, PLC delta 3 was less sensit ive to Ca2+ stimulation. This is in contrast to PLC delta 1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposo me assays. We conclude that Ca2+ is necessary to induce specific confo rmational changes of PLC delta 3, which leads to a productive orientat ion of the catalytic domain relative to the membrane. The regulatory p roperties of PLC delta 3 described in this report suggest that PLC del ta 3 has a relatively low activity in cellular conditions that fully a ctivate PLC delta 1.