MEMBRANE DOMAINS CONTAINING PHOSPHATIDYLSERINE AND SUBSTRATE CAN BE IMPORTANT FOR THE ACTIVATION OF PROTEIN-KINASE-C

The relationship between lipid domains and enzyme activity was studied via the direct visualization and quantitation of domains by fluoresce nce digital imaging microscopy. The substrate used in these experiment s was a basic peptide derived from a prominent cellular substrate (MAR CKS) of protein kinase C. The MARCKS peptide and phosphatidylserine, w hich were labeled by two different fluorophores, colocalized into doma ins in large vesicles (5-10 mu m). Increasing the ionic strength disru pted the domains of the MARCKS peptide and phosphatidylserine, and thi s was accompanied by a decrease in protein kinase C activity. Dansylpo lylysine, which inhibits protein kinase C, was similar to the MARCKS p eptide in forming domains enriched in phosphatidylserine. The degree o f enrichment of the MARCKS peptide in the phosphatidylserine domains d ecreased proportionally with protein kinase C activity when polylysine was added. Polylysine caused the MARCKS peptide to be displaced from the domains into the nondomain areas of the vesicles. This suggested t hat binding of the substrate to the vesicles was not the critical fact or for protein kinase C activity, but rather it was the organization o f the substrate into domains that was related to the activation of the enzyme. Gramicidin, which was chosen to represent a neutral membrane protein, was excluded from the domains with phosphatidylserine, and it had no effect on the enrichment of the domains or the enzyme activity . The results of this study show that the formation of membrane domain s can be important for the activation of protein kinase C and the acti vity can be inhibited by disrupting the domains.