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

Abstract

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 multifunctional, having numerous protein-protein interactions with Ca 2+ -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-domaincontaining 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. CIHR Author ManuscriptCIHR Author Manuscript CIHR Author ManuscriptPreviously, 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.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

“…These minor MBP isoforms have been observed in the nucleus of primary OLG cultures and OLGs in the mouse brain [25][26][27] and are also enriched in the radial component of compact myelin [28,29]. We have found that full-length 21.5-kDa MBP was still localized primarily to the nucleus despite the presence of the 3′UTR [20][21][22]. The minor isoforms have been speculated to play key developmental roles that may regulate myelinogenesis [25,27,[30][31][32].…”

“…This cell line stains positive for both the NG2 and A2B5 antigens, which are markers for oligodendroglial progenitor cells (OPCs), and lacks expression of classic MBP and proteolipid protein mRNAs [35]. The N19-OLG line has been successfully employed in other studies, including specifically examining the effect of Golli and classic 18.5-kDa MBP over-expression on calcium homeostasis [13,20,40,41], and on the co-localization of 18.5-kDa MBP with cytoskeletal proteins and SH3-domain-containing proteins [21,22].…”

“…Previously described plasmids coding for RFP-tagged versions of 21.5-kDa MBPs possessing a 21-nt 3′UTR were used throughout these investigations, and site-directed mutagenesis was performed as previously described [20][21][22]. The resulting constructs were confirmed by sequencing (Laboratory Services Division, University of Guelph, ON) and are shown in Table 1.…”

“…1A) significantly decrease calcium influx, via voltage operated calcium channels into primary OLGs and immortalized N19-OLG cells, in contrast to Golli isoforms [20], and that stimulation with a phorbol ester (phorbol-12-myristate-13-acetate), and with IGF-1 (insulin-like growth factor-1), spatially redistributes MBP to distinct membrane 'ruffled' regions at the cell cortex, where it associates with β-and γ-actin, cortactin, and α-tubulin [21]. Additionally, we have shown that the expression of classic 18.5-kDa MBP with the constitutively-active form of Fyn-kinase causes extensive membrane elaboration, and branching complexity at the forefront of extending N19-OLG membrane processes, a phenomenon that is abolished by substituting either proline residue within its PXXP SH3-ligand consensus motif [22].…”

“…These results demonstrate that 18.5-kDa MBP participates in and/or mediates cytoskeletal protein-protein interactions at the cytoplasmic leaflet during membrane remodeling in developing OLGs, and that several of these interactions may be facilitated by SH3-ligand domain binding of MBP with other proteins. In all of these in cellulo experiments, the constructs had a fluorescent protein fused to the amino terminus, and a 21-nucleotide untranslated region (UTR) was added to the 3′-end of the gene to ensure proper trafficking of the 18.5-kDa isoforms to the cell periphery [20][21][22][23][24].…”