Abstract
Phosphorylation of the large and small isoforms of myelin-associated glycoprotein (L- and S-MAG) was investigated in primary oligodendrocyte cultures and in immortalized Schwann cells by incubating the cells with inorganic [32P]phosphate and immunoprecipitating MAG. In oligodendrocytes, both L- and S-MAG were phosphorylated, but L-MAG was much more heavily labeled. In Schwann cells, most of the phosphorylation was in S-MAG, which is the predominant isoform expressed by these cells. In both types of cells, the principal phosphorylated amino acid in MAG was serine. Radioactive phosphothreonine and phosphotyrosine were also detected in the MAG from oligodendrocytes. In Schwann cells, there was less phosphorylation of threonine and labeled phosphotyrosine was not detected. In both oligodendrocytes and Schwann cells, the phosphorylation of MAG was stimulated by phorbol ester and a calcium ionophore, but not by forskolin. The results indicate that the phosphorylation of MAG is catalyzed by protein kinase C and possibly other calcium-activated kinases in both types of myelinating cells, but not by cAMP-activated kinase. An inhibitor of tyrosine phosphatase, ammonium vanadate, increased the amount of radioactive phosphate in MAG several fold in both oligodendrocytes and Schwann cells. However, even in the presence of vanadate, the great majority of radioactivity in MAG was in phosphoserine and only a small amount was in phosphotyrosine, suggesting that tyrosine phosphorylation of other proteins may indirectly increase the phosphorylation of MAG. The current status of our understanding of MAG phosphorylation is reviewed in the context of similarities and differences between our results and other reports in the literature.
Similar content being viewed by others
References
Afar D. E., Salzer J. L., Roder J., Braun P. E., and Bell J. C. (1990) Differential phosphorylation of myelin-associated glycoprotein isoforms in cell culture.J. Neurochem. 55, 1418–1426.
Agrawal H. C., Noronha A. B., Agrawal D., and Quarles R. H. (1990) The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system.Biochem. Biophys. Res. Commun. 169, 953–958.
Althaus H. H., Schroter J., Spoerri P., Schwartz P., Kloppner S., Rohmann, A., and Neuhoff V. (1991) Protein kinase C stimulation enhances the process formation of adult oligodendrocytes and induces proliferation.J. Neurosci. Res. 29, 481–489.
Arquint M., Roder J., Chia L. S., Down J., Wilkinson D., Bayley H., Braun P., and Dunn R. (1987) Molecular cloning and primary structure of myelin-associated glycoprotein.Proc. Natl. Acad. Sci. USA 84, 600–604.
Asotra K. and Macklin W. B. (1993) Protein kinase Cactivity modulates myelin gene expression in enriched oligodendrocytes.J. Neurosci. Res. 34, 571–588.
Bambrick L. L. and Braun P. E. (1991) Phosphorylation of myelin-associated glycoprotein in cultured oligodendrocytes.Dev. Neurosci. 13, 412–416.
Edwards A. M., ARquint M., Braun P. E., Roder J. C., Dunn R. J., Pawson T., and Bell J. C. (1988) Myelin-associated glycoprotein, a cell adhesion molecule of oligodendrocytes, is phosphorylated in brain.Mol. Cell Biol. 8, 2655–2658.
Edwards A. M., Braun P. E., and Bell J. C. (1989) Phosphorylation of myelin-associated glycoprotein in vivo and in vitro occurs only in the cytoplasmic domain of the large isoform.J. Neurochem. 52, 317–320.
Fey E. G., Wan K. M., and Penman S. (1984) Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.J. Cell Biol. 98, 1973–1984.
Frail D. E. and Braun P. E. (1984) Two developmentally regulated messenger RNAs differing in their coding region may exist for the myelin-associated glycoprotein.J. Biol. Chem. 259, 14,857–14,862.
Goda S. and Quarles R. H. (1990) Expression of MAG in a line of immortalized Schwann cells.Trans. Amer. Soc. Neurochem. 21, 240.
Goda S., Hammer J., Kobile D., and Quarles R. H. (1991) Expression of the myelin-associated glycoprotein in cultures of immortalized Schwann cells.J. Neurochem. 56, 1354–1361.
Inuzuka T., Fujita N., Sato S., Baba H., Nakano R., Ishiguro H., and Miyatake T. (1991) Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system.Brain Res. 562, 173–175.
Jaramillo M. L., Afar D. E., Almazan G., and Bell J. C. (1994) Identification of tyrosine 620 as the major phosphorylation site of myelin-associated glycoprotein and its implication in interacting with signaling molecules.J. Biol. Chem. 269, 27,240–27,245.
Johnson D. and Quarles R. H. (1986) Deposition of the myelin-associated glycoprotein in specific regions of the developing rat central nervous system.Brain Res. 393, 263–266.
Johnson D., Quarles R. H., and Brady R. O. (1982) A radioimmunoassay for the myelin-associated glycoprotein.J. Neurochem. 39, 1356–1362.
Kirchhoff F., Hofer H. W., and Schachner M. (1993) Myelin-associated glycoprotein is phosphorylated by protein kinase C.J. Neurosci. Res. 36, 368–381.
Lai C., Brow M. A., Nave K. A., Noronha A. B., Quarles R. H., Bloom F. E., Milner R. J., and Sutcliffe J. G. (1987) Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing.Proc. Natl. Acad. Sci. USA 84, 4337–4341.
McCarthy K. D. and de Vellis J. (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.J. Cell Biol. 85, 890–902.
McMorris F. A., Smith T. M., DeSalvo S., and Furlanetto R. W. (1986) Insulin-like growth factor I/somatomedin C: a potent inducer of oligodendrocyte development.Proc. Natl. Acad. Sci. USA 83, 822–826.
Peden K. W., Charles C., Sanders L., and Tennekoon G. I. (1989) Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters.Exp. Cell Res. 185, 60–72.
Pedraza L., Frey A. B., Hempstead B. L., Colman D. R., and Salzer J. L. (1991) Differential expression of MAG isoforms during development.J. Neurosci. Res. 29, 141–148.
Porter S., Glaser L., and Bunge R. P. (1987) Release of autocrine growth factor by primary and immortalized Schwann cells.Proc. Natl. Acad. Sci. USA 84, 7768–7772.
Quarles R., Colman D., Salzer J., and Trapp B. (1992) Myelin-associated glycoprotein: structure-function relationships and involvement in neurological diseases, inMyelin: Biology and Chemistry (Martenson R., ed.), CRC, Boca Raton, FL, pp. 4413–4448.
Quarles R. H., Barbarash G. R., and MacIntosh T. D. (1985) Methods for the identification and characterization of glycoproteins in central and peripheral myelin, inResearch Methods in Neurochemistry, vol. 6 (Marks N. and Rodnight R., eds.), Plenum, New York, pp. 303–357.
Quarles R. H., Hammer J. A., and Trapp B. D. (1990) The immunoglobulin gene superfamily and myelination.Prog. Clin. Biol. Res. 336, 49–79.
Quarles R. H., Johnson D., Brady R. O., and Sternberger N. H. (1981) Preparation and characterization of antisera to the myelin-associated glycoprotein.Neurochem. Res. 6, 1115–1127.
Salzer J. L., Holmes W. P., and Colman D. R. (1987) The amino acid sequences of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily.J. Cell Biol. 104: 957–965.
Trapp B. D., Andrews S. B., Cootauco C., and Quarles R. (1989) The myelin-associated glycoprotein is enriched in multivesicular bodies and periaxonal membranes of actively myelinating oligodendrocytes.J. Cell Biol. 109, 2417–2426.
Trapp B. D. and Quarles R. H. (1982) Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin.J. Cell Biol. 92, 877–882.
Tropak M. B., Johnson P. W., Dunn R. J., and Roder J. C. (1988) Differential splicing of MAG transcripts during CNS and PNS development,Brain Res. 464, 143–155.
Umemori H., Sato S., Yagi T., Aizawa S., and Yamamoto T. (1994) Initial events of myelination involve Fyn tyrosine kinase signaling.Nature 367, 572–576.
Yim S. H., Hammer J. A., and Quarles R. H. (1993) Phosphorylation of MAG in control and GM3-treated oligodendrocytes.Trans. Amer. Soc. Neurochem. 24, 223.
Yim S. H., Farrer R. G., Hammer J. A., Yavin E., and Quarles R. H. (1994) Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside.J. Neurosci. Res. 38, 268–281.
Yim S. H. and Quarles R. H. (1992) Biosynthesis and expression of the myelin-associated glycoprotein in cultured oligodendrocytes from adult bovine brain.J. Neurosci. Res. 33, 370–378.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Yim, S.H., Toda, Ki., Goda, S. et al. Comparison of the phosphorylation of myelin-associated glycoprotein in cultured oligodendrocytes and schwann cells. J Mol Neurosci 6, 63–74 (1995). https://doi.org/10.1007/BF02736760
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02736760