Diacylglycerol-induced membrane targeting and activation of protein kinase Cε -: Mechanistic differences between protein kinases Cδ and Cε

Two novel protein kinases C (PKC), PKC delta and PKC epsilon, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)- mediated membrane binding of PKC epsilon and compared it with that of PKC delta. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKC epsilon have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKC epsilon mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKC epsilon. The C1 domains of PKC epsilon are not conformationally restricted and readily accessible for DAG binding unlike those of PKC delta. Consequently, phosphatidylserine-dependent unleashing of C1 domains seen with PKC delta was not necessary for PKC epsilon. Cell studies with fluorescent protein-tagged PKCs showed that, due to the lack of lipid headgroup selectivity, PKC epsilon translocated to both the plasma membrane and the nuclear membrane, whereas PKC delta migrates specifically to the plasma membrane under the conditions in which DAG is evenly distributed among intracellular membranes of HEK293 cells. Also, PKC epsilon translocated much faster than PKC delta due to conformational flexibility of its C1 domains. Collectively, these results provide new insight into the differential activation mechanisms of PKC delta and PKC epsilon based on different structural and functional properties of their C1 domains.