REGULATION OF PHOSPHOLIPASE-C DELTA-1 BY SPHINGOSINE

Sphingosine, which is on the pathway of sphingomyelin degradation, act ivates phospholipase C (PLC) delta 1 moderately. In the liposome assay effect of sphingosine on PLC delta 1 activity depends on KCl concentr ation. Stimulation of PLC delta 1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhib ition of PLC delta 1 by sphingomyelin; To determine the domain of PLC delta 1 which interacts with sphingosine active proteolytic fragments of PLC delta 1 were generated by trypsin digestion of the native enzym e. Sphingosine affects the activity of PLC delta 1 fragment which lack ed the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of P LC delta 1. These observations indicate that for interaction of sphing osine with PLC delta 1 intact domain that span regions of conservation , designated as X and Y is necessary. When the activity of PLC delta 1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphin gosine strongly inhibited PLC delta 1. The other homolog of sphingosin e 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta 1 to muc h lesser extent. The activity of PLC delta 1 was inhibited by 68% and 22% in the presence of 20 mu M sphingosine and phytosphingosine, respe ctively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta 1 in the cell.