The long term goals are to contribute to an understanding of the roles that the cellular lipids and their breakdown products play in cellular regulation. Lipids play profound and complex roles in the regulation of members of the protein kinase C (PKC) family. The proposed studies seek to elucidate the molecular mechanisms by which a variety of lipids regulate PKC activity as essential phospholipid cofactors, as specific activators or as inhibitors. During the last grant period, biochemical and molecular biological experiments, provided molecular insight into the exquisite specificity of PKC-lipid interactions, the domains where these interactions occur, and suitable systems for molecular analysis of individual family members. Three or more distinct segments within PKC are known to interact with lipids. The lipid-protein interaction of these segments will be elucidated by developing suitable expression systems for each segment, scaling these up to purify sufficient quantities for biochemical and structural analysis. The lipid-protein interaction within the phorbol ester binding segments, cys1 and cys2 and in C2, the calcium phospholipid binding domain (Ca1B), will be investigated using mixed micellar and vesicular techniques by employing PS and a number of analogues and other phospholipids. The structure of these peptides will be investigated by physical methods including CD and NMR in the presence and absence of lipids. Collaborative crystallography analyses are also planned. The role of the two tightly bound zinc ions within the cys1 and cys2 regions on structure and function will be investigated. Residues critical for zinc ligation will be inferred by mutational analysis and determined by NMR studies. Studies on mutants created within the cys1, cys2, and Ca1B domains seek to define segments or individual residues critically involved in lipid regulation, e.g., residues involved in stereospecific interaction with PS and DAG/phorbol esters. Studies on the role of PKC in cellular regulation, transformation, and tumor formation will be advanced by the creation of constitutively active forms of PKC which are stably expressed. These studies will contribute to the molecular mechanism of PKC regulation by lipids in signal transduction processes including cell growth. The studies will address the role of PKC in cellular transformation. The studies will likely contribute to an understanding of other diseases, cardiovascular, inflammatory, airway (asthma), dermatological (psoriasis), diabetes, and CNS where PKC is know to play critical roles.
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