| Protein kinase C (PKC) is a critical component of many signaling pathways. As such, PKC regulates such diverse phenomena as memory and learning, immune system responses, and cancer. Binding to membranes regulates the activity of PKC, and the C1 domain of PKC binds to membranes in response to the lipid second messenger, diacylglycerol (DG). This dissertation describes the molecular nature by which the C1 domain of PKC interacts with membranes. First, a fluorescent reporter is used with stopped-flow fluorescent spectroscopy to analyze the binding kinetics between the C1 domain and lipid vesicles. Kinetic data reveal that both specific and non-specific interactions guide the association of the C1 domain with membranes, whereas hydrophobic contacts between the C1 domain and its ligand determine the rate of dissociation. The data provide evidence for a model that describes the mechanism by which the C1 domain binds to membranes and explain how the DG-mimicking, tumor-promoting phorbol esters (i.e., PMA) exert their potency. Second, an evolutionarily-conserved residue that regulates the C1 domain's affinity for DG has been identified. This residue, which is a conservative change from tryptophan to tyrosine, lies at the apex of the ligand-binding pocket and lowers the affinity for DG 30-fold without affecting the affinity for PMA; tryptophan at this position also localizes the isolated domain to internal membranes. Molecular modeling suggests that this amino acid regulates the width of the ligand-binding pocket, and the identity of this residue explains the Golgi localization of novel PKC isoforms and why novel PKC isoforms rely solely on their C1 domains for activation. Finally, modeling of the atypical C1 domain of PKCzeta reveals that the C1 domain cannot bind DG or PMA due to a cluster of basic residues occluding the binding pocket. Moreover, this domain does not have a higher intrinsic affinity for a variety of phospholipids than does a typical C1 domain, despite having a more positive electrostatic potential. The data presented in this dissertation redefine the molecular nature of the interaction of the C1 domain with lipid membranes and offer insights into this unique class of lipid-targeting domains. |
