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

Abstract

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.

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

“…The deiminated 18.5-kDa C8 form is increased in myelin from children and also from adult multiple sclerosis patients [85], suggesting that it could have both physiological developmental and pathological roles. Although the deiminated form does not bind actin filaments, microtubules, and the SH3-domain of Fyn to a membrane surface as well as the unmodified 18.5-kDa C1 form [23,34,86], here it colocalized similarly with other proteins in PMA-stimulated N19-OLGs as the unmodified form did. The full-length classic 21.5-kDa isoform redistributed to the cytosol in PMAstimulated N19-OLGs and also co-localized with actin, tubulin, and cortactin in membrane ruffles.…”

“…These studies support an important role for interaction of MBP with the cytoskeleton in OLG development and myelination [31][32][33]. The 18.5-kDa MBP splice isoform has also been shown to bind the SH3-domain of Fyn to lipid vesicles [34]. To date, these in vitro biochemical techniques have added to our understanding of MBP's interactions with other proteins in vitro.…”

“…resulting in net charge reduction modulate these diverse interactions. Classic MBP isoforms also have a PXXP motif which is a ligand for SH3-domains, and have been shown to bind to several proteins with SH3-domains and tether the SH3-domain of Fyn to a membrane surface in vitro [16,[34][35][36]. The phosphorylated form of MBP is enriched along with cytoskeletal proteins and kinases including MAPK, Fyn, and Lyn within insoluble Triton-X100 membrane domains when extracted from myelin [67,69,70].…”

“…Phosphorylation protects the protein from proteases such as trypsin [80], and has potential structure-stabilizing effects [74,81,82]. The MAPK phosphorylation of MBP decreases its ability to bundle actin filaments and to bind actin filaments, microtubules, and the SH3-domain of Fyn to a lipid membrane [26,34,83]. Phosphorylation at these sites may induce a conformational change of this segment of the protein and its disposition with respect to the membrane surface [74,75].…”

Glyphosate pathways to modern diseases VI: Prions, amyloidoses and autoimmune neurological diseases

“…It assembles actin filaments and microtubules, binds actin filaments and SH3 domains to membrane surfaces, and participates in signal transduction in oligodendrocytes and myelin. A central proline-rich region in MBP is functionally significant [108][109][110] and, in particular, is a binding site for Fyn-SH3, a key regulatory protein [111]. Proline substitutions of the SH3 ligand decrease its affinity for the Fyn-SH3 domain [108].…”