Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelin.
ABSTRACT To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (M(r) 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP-MBP-S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP-MBP-S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin-axonal interactions.
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ABSTRACT: Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly wrapped multilamellar structure around neurons enabling rapid and efficient signal propagation. The gene of oligodendrocyte lineage (golli) gives rise to a variety of developmentally regulated splice isoforms of myelin basic protein (MBP), denoted golli for early forms and classic for later ones. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa; its central role is to maintain the structural integrity of the myelin sheath, by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane in a tight, spiral, multilamellar arrangement. This protein's extreme physicochemical properties, net charge of +19 at neutral pH, low proportion of hydrophobic residues, alternating regions of predicted intrinsic disorder and order, induced folding upon association with membranes and other proteins, and diversification via combinatorial post-translational modifications, define not only its role as a molecular Velcro in compact myelin, but as a multifunctional hub that may also bind to a number of other proteins and small molecule ligands in myelinating oligodendrocytes. In particular, MBP may link the underlying cytoskeleton and proteins containing SH3 domains to the membrane, allowing it to transduce transmembrane signals to the cytosol. These associations are facilitated by MBP being an intrinsically disordered protein, creating a large effective protein surface, and by the formation of transient and/or induced ordered secondary structure elements for molecular recognition. These processes can be modulated by a molecular barcode of numerous post-translational modifications and interactions with proteins such as calmodulin. In the human demyelinating disease multiple sclerosis, an aberrant pattern of modifications may contribute to demyelination and confound inherent attempts at repair. The conformational dynamics of the various isoforms and modified variants of MBP and their interactions with other proteins potentially allow them to participate in events coupling extracellular signals to cytoskeletal organization during myelination or remyelination. Various biophysical and cell biological approaches are beginning to elucidate these properties of MBP and are leading to a new understanding of the role of this protein as a linker and/or hub in structural and signaling networks in oligodendrocytes and myelin.Biochemistry 07/2009; 48(34):8094-104. · 3.42 Impact Factor
Article: Effect of phosphorylation of myelin basic protein by MAPK on its interactions with actin and actin binding to a lipid membrane in vitro.[show abstract] [hide abstract]
ABSTRACT: Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isomers of varying charge, some resulting from phosphorylation at several sites by different kinases, including mitogen-activated protein kinase (MAPK). Phosphorylation of MBP in oligodendrocytes occurs in response to various extracellular stimuli. Phosphorylation/dephosphorylation of MBP also occurs in the myelin sheath in response to electrical activity in the brain. Here we investigate the effect of phosphorylation of MBP on its interaction with actin in vitro by phosphorylating the most highly charged unmodified isomer, C1, at two sites with MAPK. Phosphorylation decreased the ability of MBP to polymerize actin and to bundle actin filaments but had no effect on the dissociation constant of the MBP-actin complex or on the ability of Ca2+-calmodulin to dissociate the complex. The most significant effect of phosphorylation on the MBP-actin complex was a dramatic reduction in its ability to bind to negatively charged lipid bilayers. The effect was much greater than that reported earlier for another charge isomer of MBP, C8, in which six arginines were deiminated to citrulline, resulting in a reduction of net positive charge of 6. These results indicate that although average electrostatic forces are the primary determinant of the interaction of MBP with actin, phosphorylation may have an additional effect due to a site-specific electrostatic effect or to a conformational change. Thus, phosphorylation of MBP, which occurs in response to various extracellular signals in both myelin and oligodendrocytes, attenuates the ability of MBP to polymerize and bundle actin and to bind it to a negatively charged membrane.Biochemistry 02/2006; 45(2):391-401. · 3.42 Impact Factor