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 |
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Authors: | Graham S T Smith Lopamudra Homchaudhuri Joan M Boggs George Harauz |
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Institution: | (1) Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada;(2) Molecular Structure and Function Program, Hospital for Sick Children, Toronto, ON, Canada;(3) Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; |
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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 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. |
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