title: The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition.
abstract: Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) 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 PIP(2). 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 PIP(2), 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 PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.
authors: Instituto Murciano de Investigaciones Biomédicas, Regional Campus of International Excellence "Campus Mare Nostrum", Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain.
authors: Angel
authors: A
authors: Pérez-Lara
authors: Antonio L
authors: AL
authors: Egea-Jiménez
authors: Alessio
authors: A
authors: Ausili
authors: Senena
authors: S
authors: Corbalán-García
authors: Juan C
authors: JC
authors: Gómez-Fernández
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013552:Swine
chemical_list: D004075:Diglycerides; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D010718:Phosphatidylserines; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; D051571:Protein Kinase C-alpha; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; C013965:diolein