Proline substitutions and threonine pseudophosphorylation of the SH3 ligand of 18.5‐kDa myelin basic protein decrease its affinity for the Fyn‐SH3 domain and alter process development and protein localization in oligodendrocytes

Abstract

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function.

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].…”

“…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.…”

“…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.…”

“…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].…”