Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Neurochem Res. 2012 Jun;37(6):1277-95. doi: 10.1007/s11064-011-0700-2. Epub 2012 Jan 17.
The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.
18.5kDa 经典髓鞘碱性蛋白（MBP）是一种源自 Golli（少突胶质细胞谱系基因）基因复合物的无规则结构蛋白，负责中枢神经系统髓鞘的压缩。这种 MBP 剪接异构体还具有多种翻译后修饰，包括磷酸化、脱亚氨基化、甲基化和脱酰胺化，这些修饰降低了其总净电荷，并改变了其在少突胶质细胞（OLG）中的蛋白质和脂质相互作用。最初认为 MBP 只是髓鞘的结构成分；然而，进一步的研究表明，MBP 具有多种功能，与 Ca²⁺-钙调蛋白、肌动蛋白、微管蛋白和具有 SH3 结构域的蛋白质有许多蛋白-蛋白相互作用，并可以在体外将这些蛋白质固定在脂质膜上。在这里，我们使用转染了荧光标记的 MBP、肌动蛋白、微管蛋白和紧密连接蛋白 1（ZO-1）的早期发育 N19-OLG 体内研究了经典 18.5kDa MBP 的细胞骨架相互作用。我们表明，MBP 重新分布到质膜的独特“膜皱襞”区域，在质膜皱襞区域与肌动蛋白和微管蛋白以及含有 SH3 结构域的蛋白质 cortactin 和 ZO-1 共定位，当用 PMA 刺激时，PMA 是蛋白激酶 C 途径的有效激活剂。此外，使用磷酸化特异性抗体染色，我们显示在膜皱襞 OLG 中 Thr98 磷酸化的 MBP 增加。先前的研究表明，MBP 的 Thr98 磷酸化会影响其构象、与其他蛋白质的相互作用以及体外将其他蛋白质固定在膜上。在这里，在细胞在层粘连蛋白-2 上生长时，IGF-1 刺激诱导的新焦点粘附接触中，MBP 和肌动蛋白也共定位。这项研究支持经典 MBP 异构体在 OLG 中质膜和细胞骨架重塑过程中的细胞骨架和其他蛋白质-蛋白质相互作用中的作用。
