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 fluorescence digital imaging microscopy. The substrate used in these experiments was a basic peptide derived from a prominent cellular substrate (MARCKS) of protein kinase C. The MARCKS peptide and phosphatidylserine, which were labeled by two different fluorophores, colocalized into domains in large vesicles (5-10 mu m). Increasing the ionic strength disrupted the domains of the MARCKS peptide and phosphatidylserine, and this was accompanied by a decrease in protein kinase C activity. Dansylpolylysine, which inhibits protein kinase C, was similar to the MARCKS peptide in forming domains enriched in phosphatidylserine. The degree of enrichment of the MARCKS peptide in the phosphatidylserine domains decreased 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 that binding of the substrate to the vesicles was not the critical factor for protein kinase C activity, but rather it was the organization of 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 domains can be important for the activation of protein kinase C and the activity can be inhibited by disrupting the domains.