title: Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.
abstract: Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface.
authors.affiliation: Biomolecular NMR Laboratory, Organic Chemistry Department, University of Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain; Institute for Research in Biomedicine (IRB-Barcelona), Baldiri Reixac 10-12, 08028 Barcelona, Spain.
authors.forename: Anabel-Lise
authors.initials: AL
authors.lastname: Le Roux
authors.affiliation: Department of Physicochemistry, Faculty of Pharmacy, Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Joan XXIII 27-31, 08028 Barcelona, Spain.
authors.forename: Maria Antònia
authors.initials: MA
authors.lastname: Busquets
authors.affiliation: Department of Physical Chemistry, Faculty of Chemistry, Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain.
authors.forename: Francesc
authors.initials: F
authors.lastname: Sagués
authors.affiliation: Biomolecular NMR Laboratory, Organic Chemistry Department, University of Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain. Electronic address: mpons@ub.edu.
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; C002773:dimyristoylphosphatidylglycerol; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D004134:Dimyristoylphosphatidylcholine
keywords: Antibody capture; Cell signaling; Intrinsically disordered proteins (IDP); Myristoylated proteins; Protein dimerization on membranes; SH4 domains; Surface plasmon resonance; c-Src; c-Src trafficking