Modes of SH3-Domain Interactions of 18.5 kDa Myelin Basic Protein IN Vitro and in Oligodendrocytes

Abstract

The developmentally-regulated myelin basic proteins (MBPs) play key roles in central nervous system (CNS) myelin formation by oligodendrocytes. They are highly positively-charged, intrinsically disordered, multifunctional proteins having several alternatively-spliced isoforms and combinatorial post-translational modifications. The most common 18.5kDa MBP isoform contains a proline-rich region (murine sequence T92PRTPPPS99) which comprises a minimal SH3-ligand. We have previously shown that 18.5 kDa MBP binds to Fyn, a member of the Src family of tyrosine kinases involved in signaling pathways during CNS development. Here, we have produced an isotopically-labeled fragment of MBP which contains the proline-rich region, and are studying the mode of interaction between MBP and the SH3 domain of Fyn through solution NMR spectroscopy and isothermal titration calorimetry (ITC). We have constructed MBP variants pseudo-phosphorylated at T92 and T95, and with P93G and P96G substitutions to disrupt the conformation of the SH3-ligand. Using ITC, these mutant MBPs are shown to have a decreased affinity towards the SH3-domain of Fyn. Solution NMR spectroscopy shows altered chemical shift patterns for the different variants, confirming that this segment is indeed the SH3-target and that the association can be modulated by phosphorylation. Concomitant over-expression of complementary GFP-tagged MBP forms in cultured oligodendrocytes results in aberrant elongation of membrane processes, increased branching complexity, and in some cases, trafficking of MBP to the nucleus. These data as a whole indicate that MBP's SH3-ligand domain plays a key role in intracellular protein interactions and may be required for proper membrane elaboration in myelin.Acknowledgements: Funded by the Canadian Institutes for Health Research (JMB, GH), NSERC Canada (GH), and by Doctoral Studentships from the Multiple Sclerosis Society of Canada (MDA, GSTS).