The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition

Protein kinase C alpha (PKC alpha) is activated by its translocation to the membrane. Activity assays show the importance of PIP2 in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP2. The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP2, the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP2. We propose a model in which PKC alpha binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKC alpha driven by interactions with POPS and/or PIP2 although interactions with DOG are involved too. PKC alpha searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands. (c) 2012 Elsevier B.V. All rights reserved.