Structural and Dynamical Basis of Protein Kinase C Alpha Regulation by the C-Terminal Tail

Abstract

Protein Kinase C (PKC) isoenzymes are dynamic multi-modular proteins that regulate signal transduction processes at the membrane surface. When activated by second messengers, such as Ca2+ and diacylglycerol, PKC undergoes a drastic conformational transition from the inactive cytosolic state to the activated membrane-bound state. The structure of either state of PKC is not known. Using NMR, we demonstrate that the isolated Ca2+-sensing lipid-binding C2 domain of PKCα interacts with the C-terminal tail of the kinase. We determined the structure of the complex between the C2 domain and the peptide corresponding to the phosphorylated hydrophobic motif of the C-terminal tail. The structure shows that the hydrophobic motif binds to the conserved lysine-rich cluster region of C2. The interface is stabilized by electrostatic interactions and stacking of the aromatic sidechains. We validated our structural model by mutating the interacting residues in full-length PKC, and characterizing these variants using in vitro FRET experiments and FRET-detected membrane translocation experiments in live cells. In addition, NMR-detected binding studies revealed that the hydrophobic motif and Ca2+ synergistically enhance each other's affinities to C2. We propose a model where the C-terminal tail plays a dual role in PKC regulation: auto-inhibitory, through its interaction with the lysine-rich cluster of the C2 domain; and activating, through sensitization of PKC to intracellular Ca2+ oscillations. Support: Welch Foundation grant A-1784 (TII), NSF CAREER award CHE-1151435 (TII), NIH GM108998 (TII), and NIH GM43154 (ACN).