PROTEIN KINASE C--IN VITRO STUDIES

蛋白激酶C （PKC）被认为是一个关键的调控酶

Protein kinase C (PKC) is a key regulatory enzyme believed to be involved in many cellular processes such as cell growth and differentation, hormone release, platelet activation, and many other events. Phosphorylation of substrate proteins usually requires Ca+2 and phospholipid as well as the second messenger, diacylglycerol (DAG). Biologically active phorbol esters substitute for DAG. Despite abundant current studies on this protein, little is known about the physical entity that generates kinase activity and how various activators exert their influence. We have found that the Ca+2 and the phospholipid requirements of PKC greatly exceed those needed for PKC-membrane binding and that the cofactor (phospholipid, Ca+2, and/or DAG) requirements of PKC activation are dependent on the choice of substrate. In searching for an explanation for this behavior, we observed the interaction of substrates with the phospholipid was a critical aspect of PKC action and all good in vito substrates not only bound to phospholipid but caused aggregation of phospholipid vesicles. These observations illustrate the need to evaluate all of the different interactions of PKC, its substrates and cofactors. This study will investigate the interactions of PKC and other components in various states (e.g. phospholipid vesicles and monolayers) and will determine how the events and their more detailed aspects are important to develoment of kinase activity. Identification of nonaggregated assay systems will be attempted. Metal ion binding properties of PKC will be examined by several techniques including direct binding measurements and fluorescence methods. Selectivity of PKC for membrane structural features (e.g. composition and surface curvature) will be studied to determine the factors involved in PKC-membrane binding and in generating nondissociable membrane-bound PKC. The effect of phosphorylation on protein-membrane assembly will be assessed using two different proteins, histones and myelin basic protein. The effect of Ca+2, DAG or phorbol esters on the conformation and on the dynamics of PKC-membrane assembly and dissociation will be investigated using fluorescence and CD methods. All interactions will be compared to appearance of PKC activity to help define those aspects of the enzyme-substrate- membrane-cofactor complex that are critical to development of kinase activity. Defining the in vitro properties will improve our understanding of how in vivo targets for this enzyme are identified.