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

Oligodendrocytes are specialized glial cells of the central nervous system (CNS) that develop from precursor cells and migrate throughout the spinal cord and regions of the brain during mammalian development (Bradl and Lassmann, 2010; Miron et al., 2011). They extend cellular processes that form a multilamellar insulating sheath around developing axons (Bunge et al., 1962; Bunge, 1968; Baumann and Pham-Dinh, 2001). The CNS myelin contains many essential proteins that facilitate the structure, function, and compaction of myelin, including the developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex (Campagnoni et al., 1993; Pribyl et al., 1993; Givogri et al., 2001; Jacobs et al., 2009; Fulton et al., 2010a). Although the basic proteins play a key role in myelin compaction, they are also highly positively charged, intrinsically disordered proteins having numerous alternatively spliced isoforms and combinatorial posttranslational modifications such as deimination and phosphorylation. They also have a proline-rich region comprising amino acids T92–S99 (murine sequence relative to 18.5-kDa isoform) –T92PRTPPPS99–, which contains an SH3 ligand motif (Polverini et al., 2008). These properties facilitate and regulate a variety of biological interactions with proteins such as actin, tubulin, calmodulin, and SH3 domain proteins (for review see Harauz et al., 2004, 2009; Boggs, 2006, 2008; Harauz and Libich, 2009). Our group has extensively utilized recombinant hexahistidine-tagged versions of “classical” murine MBP isoforms, such as rmMBP-C1, which emulates the predominant, minimally modified 18.5-kDa C1 component of healthy myelin (Bates et al., 2000), and 21.5-kDa MBP (Hill et al., 2005; Hill and Harauz, 2005), which is expressed earlier in development than the 18.5-kDa isoform. Utilizing purified recombinant MBPs (rmMBPs) for in vitro studies has provided insight into MBP’s multifunctionality and its interactions with actin, tubulin, Ca2+-calmodulin, and SH3 domains (Boggs et al., 2005, 2011; Hill et al., 2005; Hill and Harauz, 2005; Libich and Harauz, 2008; Polverini et al., 2008; Homchaudhuri et al., 2009; Ahmed et al., 2009; Bamm et al., 2010). Moreover, we have constructed green and red fluorescent protein-tagged (GFP- and RFP-tagged) and untagged versions of the 18.5- and 21.5-kDa classical MBP isoforms for cell transfection and investigation of MBP’s multifunctionality in cultured oligodendroglial cells (Smith et al., 2010). We have shown that these MBP isoforms decreased Ca2+ influx through voltage-operated Ca2+ channels (VOCC) in cultured N19 immortalized oligodendroglial cells (OLGs) and primary oligodendroglial progenitor cells (OPCs; Smith et al., 2011), suggesting that classical MBP plays an important role in calcium homeostasis. We have also shown increased co-localization of MBP with actin, tubulin, the SH3-domain-containing actin-remodeling protein cortactin, and the SH3-domain-containing junctional protein ZO-1, during membrane remodeling in N19-OLGs (Smith et al., 2010). For the present study, we generated further variants to examine the role of the highly conserved central segment that encompasses the putative SH3 ligand (Polverini et al., 2008, 2011; Homchaudhuri et al., 2009). Studies using recombinant murine MBP (rmMBP; Bates et al., 2000, 2002) have assessed the structural and binding properties of MBP to SH3 domains using circular dichroic spectroscopy and dot-blot microarray analyses, respectively (Polverini et al., 2008; Harauz and Libich, 2009; Homchaudhuri et al., 2009). Under physiological conditions, this region forms a poly-proline type II (PPII) conformation in vitro, and MBP bound a number of SH3 domains on a microarray, including that of Fyn (Polverini et al., 2008; Harauz and Libich, 2009). These studies also demonstrated that the PPII conformation in aqueous solution is stabilized by the phosphorylation of Thr92 and Thr95 (murine 18.5-kDa isoform sequence numbering, omitting the N-terminal methionine, which is cleaved), present within and/or adjacent to the helix. Moreover, MBP was shown to bind the Fyn-SH3 domain to lipid vesicles, and in vitro phosphorylation of MBP at Thr92 and/or Thr95 decreased this binding (Homchaudhuri et al., 2009). Molecular dynamics simulations indicate also that phosphorylation at these sites can alter the local conformation of the protein and the degree of penetration of the central membrane-anchoring segment into a lipid bilayer (Polverini et al., 2011). We have thus proposed that this region of the protein constitutes an important molecular switch (Harauz et al., 2009; Harauz and Libich, 2009; Bessonov et al., 2010; Polverini et al., 2011). Fyn is a member of the Src family of tyrosine protein-specific kinases that is localized primarily to the cytoplasmic leaflet of the OLG plasma membrane, where it can participate in a variety of different signaling pathways via integrins and Ras activation during CNS development (Manie et al., 1997; Resh, 1998) and plays an important role in OLG differentiation and myelination (for review see Kramer-Albers and White, 2011). Mouse knockouts for Fyn have shown significant MBP mRNA attenuation during the most active period of myelinogenesis (P13 and P20), resulting in hypomyelination (Biffiger et al., 2000; Lu et al., 2005). Fyn has been postulated to be a key regulatory element of the myelination process that triggers phosphorylation of hnRNPA2 (heterogeneous nuclear ribonucleoprotein A2), responsible for efficient transport of MBP mRNA to the site of glial–neuronal contact in developing OLGs (Seiwa et al., 2000, 2007; Laursen et al., 2011). Additional studies examining Fyn homologues, such as Src, have shown that members of this kinase family are involved in diverse cellular functions, including adhesion and calcium flux (Laursen et al., 2009). In this current study, we provide further evidence for the interaction of the SH3 ligand of MBP with the Fyn-SH3 domain, utilizing site-directed SH3 ligand-substituted versions of rmMBP-C1 and biophysical approaches. We also provide evidence for a physiological role of MBP’s SH3 ligand domain in cultured N19-OLGs. In particular, we have constructed recombinant murine 18.5-kDa MBP variants with Pro-to-Gly substitutions to disrupt the PXXP SH3 ligand motif or with Thr-to-Glu substitutions to mimic phosphorylation of Thr92 and Thr95 (see Fig. 1). Using isothermal titration calorimetry (ITC), we show that these variants have a decreased affinity for the Fyn-SH3 domain. Overexpression of GFP-tagged variants in cultured N19-OLGs produced aberrant elongation of membrane processes, increased branching complexity, and in some cases caused the 18.5-kDa MBP isoform to traffic to the nucleus. Furthermore, these substitutions to the SH3 ligand domain inhibited the ability of MBP to decrease L-type VOCC-mediated calcium influx. Coexpression of MBP with a constitutively active form of Fyn kinase caused a remarkable increase in elaboration of membrane processes and stimulated development of N19-OLGs. Substitutions of the SH3 ligand domain of MBP prevented this effect, indicating that interaction of MBP with an SH3 domain protein, most likely Fyn, is responsible for this effect. These results suggest that MBP’s SH3 ligand domain may play a key role in interactions with signaling proteins such as Fyn at the cytoplasmic surface of the plasma membrane that may be required for membrane elaboration of OLGs. Fig. 1 A: Schematic of recombinant 18.5-kDa MBP variants used for isothermal titration calorimetry. B: Schematic of the MBP-C1 and SH3 ligand mutagenic constructs produced for immortalized N19/N20.1-OLG cell culture studies. The GFP has been fused to the N-terminus ...