Differential Membrane-Binding and Activation Mechanisms of Protein Kinase C-α and -ε

Abstract

To elucidate the mechanisms of membrane binding and activation of conventional and novel protein kinase C (PKC), we measured the interactions of rat PKC-α and -ε with phospholipid monolayers and vesicles of various compositions. Besides the established difference in calcium requirement, the two isoforms showed major differences in their membrane-binding and activation mechanisms. For PKC-α, diacylglycerol (DG) specifically enhanced the binding of PKC-α to phosphatidylserine (PS)-containing vesicles by 2 orders of magnitude, allowing PKC-α high specificity for PS. Also, PKC-α could penetrate into the phospholipid monolayer with a packing density comparable to that of the cell membrane only in the presence of Ca²⁺ and PS. When compared to PKC-α, PKC-ε had lower binding affinity for PS-containing vesicles both in the presence and in the absence of DG. As a result, PKC-ε did not show pronounced specificity for PS. Also, PKC-ε showed reduced penetration into PS-containing monolayers, which was comparable to the Ca²⁺-independent penetration of PKC-α into the same monolayers. Taken together, these results suggest the following: (1) The role of Ca²⁺ in the membrane binding of PKC-α is to expose a specific PS-binding site. (2) Once bound to membrane surfaces, PS specifically induces the partial membrane penetration of PKC-α that allows its optimal interactions with DG, hence the enhanced membrane binding and activation. (3) PKC-ε, due to the lack of Ca²⁺ binding, cannot specifically interact with PS and DG, which implies the presence of other physiological activator(s) for this isoform.