The origin of C1A-C2 interdomain interactions in protein kinase Cα

The regulatory domain of protein kinase C alpha (PKC alpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKC alpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKC alpha revealed a highly complementary interface that comprises Asp(55) - Arg(252) and Arg(42) - Glu(282) ion pairs and a Phe(72) Phe(255) aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKC alpha, supporting their involvement in interdomain interactions. In particular, D55A ( or D55K) and R252A ( or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/ R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKC alpha C1A- C2 interdomain interactions and the mechanism of lipid-mediated PKC alpha activation.