FORMATION OF MEMBRANE DOMAINS DURING THE ACTIVATION OF PROTEIN-KINASE-C

The lateral membrane organization of phosphatidylserine, diacylglycero l, substrate, and Ca2+-dependent protein kinase C in large unilamellar vesicles was investigated by using fluorescence digital imaging micro scopy. The formation of phosphatidylserine domains could be induced by either Ca2+, the MARCKS peptide, or protein kinase C. However, only C a2+ could induce diacylglycerol to partition into the phosphatidylseri ne domains. In the complete protein kinase C assay mixture, two separa te triple-labeling experiments demonstrated the colocalization of phos phatidylserine, protein kinase C, diacylglycerol, and the MARCKS pepti de in domains, The amounts of all the labeled components in whole vesi cles and in domains were measured at various concentrations of either phosphatidylserine, Ca2+, diacylglycerol, or the MARCKS peptide or wit h the addition of polylysine. The role of each component in forming me mbrane domains and in mediating the enzyme activity was analyzed. The results indicated that the inclusion of the MARCKS peptide in the doma ins, not just the binding of the substrate to vesicles, was especially important for PKC activity. The formation of PKC domains required the presence of DAG and Ca2+ at physiological ionic strength. The PKC act ivity was proportional to the amounts of PKC and substrate in the doma ins. The results also showed that the MARCKS peptide left the domains after being phosphorylated. A model for the activation of protein kina se C involving sequestering of the reaction components into membrane d omains is proposed. The efficiency of the reaction was greatly increas ed by concentrating the activators, the enzyme, and the substrate into domains.