Influence of membrane surface charge and post-translational modifications to myelin basic protein on its ability to tether the Fyn-SH3 domain to a membrane in vitro.
Myelin basic protein (MBP) is a highly post-translationally modified, multifunctional structural component of central nervous system myelin, adhering to phospholipid membranes and assembling cytoskeletal proteins, and has previously been shown to bind SH3 domains in vitro and tether them to a membrane surface [Polverini, E., et al. (2008) Biochemistry 47, 267-282]. Since molecular modeling shows that the Fyn-SH3 domain has a negative surface charge density even after binding the MBP ligand, we have investigated the influence of negative membrane surface charge and the effects of post-translational modifications to MBP on the interaction of the Fyn-SH3 domain with membrane-associated MBP. Using a sedimentation assay with multilamellar vesicles consisting of neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol (PI), we demonstrate that increasing the negative surface charge of the membrane by increasing the proportion of PI reduces the amount of Fyn-SH3 domain that binds to membrane-associated MBP, due to electrostatic repulsion. When one of the phosphoinositides, PI(4)P or PI(4,5)P(2) was substituted for PI in equal proportion, none of the Fyn-SH3 domain bound to MBP under the conditions that were used. Post-translational modifications of MBP which reduced its net positive charge, i.e., phosphorylation or arginine deimination, increased the degree of repulsion of Fyn-SH3 from the membrane surface, an effect further modulated by the lipid charge. This study suggests that changes in membrane negative surface charge due to protein or lipid modifications, which could occur during cell signaling, can regulate the binding of the Fyn-SH3 domain to membrane-associated MBP and thus could regulate the activity of Fyn at the oligodendrocyte membrane surface.
Homchaudhuri
Lopamudra
L
Department of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Polverini
Eugenia
E
Gao
Wen
W
Harauz
George
G
Boggs
Joan M
JM
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D051379:Mice; D008958:Models, Molecular; D004676:Myelin Basic Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009836:Oligodendroglia / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018909:src Homology Domains
D004676:Myelin Basic Protein; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn