UC San Diego

Protein kinase C (PKC) has a central role in responding to signals that cause lipid hydrolysis leading to both short- and long-term cellular responses. Membrane binding plays a key role in the activation of PKC. During activation, the regulatory C2 domain of the conventional PKCs target the kinase to membranes in response to elevated intracellular Ca²⁺. Once the C2 domain poises the enzyme at the membrane , it can engage a second membrane-targeting module, the C1 domain, by binding to the second messenger diacylglycerol. These interactions provide the energy for an activating conformational change in which the pseudosubstrate sequence is released from its binding cavity of PKC, allowing substrate binding and phosphorylation. In this dissertation, the kinetics and mechanism of the critical interaction between the C2 domain and membrane were examined. Combining biophysical, biochemical, and cellular biology techniques, the roles of electrostatic and hydrophobic interactions in the kinetics and mechanism of C2 domain membrane binding and its effects on kinase activity were elucidated. These studies show that electrostatic interactions of specific residues play a role in the retention of the C2 domain at the membrane, while hydrophobic interactions of specific residues play roles in both the membrane recruitment and retention of the C2 domain. In addition, studies with varying ionic strength and Ca²⁺ have given further insights into the role of these determinants in C2 domain membrane binding. Comparative studies of the conventional PKC isoforms and their mutants further elucidate the binding kinetics and mechanism of C2 domain binding. These studies emphasize the importance of residue 249 (numbering of PKC conventional isoforms) in both electrostatic and hydrophobic interactions that stabilize the domain- membrane complex. Taking the studies into the context of the full-length PKC, the role of these electrostatic and hydrophobic interactions in determining the kinetics, amplitude, and duration of PKC activity in cells has been monitored using live cell imaging. The imaging illustrates the importance of these electrostatic and hydrophobic interactions translates from membrane binding to kinase activity in live cells. In summary, electrostatic and hydrophobic interactions play key roles on the membrane recruitment and retention of the C2 domain