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Myelin-associated glycoprotein is phosphorylated by protein kinase C
F Kirchhoff1, H W Hofer, M Schachner
1Department of Neurobiology, University of Heidelberg, Germany.
Abstract:
The myelin-associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin-forming cells and neurons. To characterize the molecular mechanisms underlying post-translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of 32P-orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol ester phorbol-12-myristoyl-13-acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2-3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [gamma-32P]ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet-derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes.
Insights
Myelin-associated glycoprotein (MAG) phosphorylation occurs on serine residues in oligodendrocytes and Schwann cells. Protein kinase C activation enhances MAG phosphorylation, but extracellular signals do not appear to trigger this modification.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Myelin-associated glycoprotein (MAG) is crucial for neural recognition and interactions between myelin-forming cells and neurons.
- Post-translational modifications, such as phosphorylation, are implicated in signal transduction following MAG recognition events.
- Understanding MAG phosphorylation mechanisms is key to deciphering neural signaling pathways.
Purpose of the Study:
- To investigate the stimuli that induce post-translational modifications of MAG, specifically 32P-orthophosphate incorporation.
- To characterize the molecular mechanisms underlying MAG phosphorylation in cultured oligodendrocytes and differentiated Schwann cells.
- To identify potential physiological triggers for MAG phosphorylation.
Main Methods:
- Studied 32P-orthophosphate incorporation into MAG in cultured oligodendrocytes and transformed differentiated Schwann cells.
- Utilized phorbol ester (PMA) to assess the role of protein kinase C (PKC) in MAG phosphorylation.
- Incubated purified MAG with PKC and [gamma-32P]ATP to confirm kinase activity.
- Exposed oligodendrocyte cultures to various extracellular signals (neuronal coculture, membrane fractions, anti-MAG antibody, PDGF) to test for phosphorylation modulation.
Main Results:
- In oligodendrocytes, both 67 and 72 kD MAG isoforms were phosphorylated exclusively on serine residues.
- In Schwann cells, only the 67 kD MAG isoform was present and phosphorylated on serine.
- Phorbol-12-myristoyl-13-acetate (PMA) enhanced MAG phosphorylation 2-3 fold, indicating PKC involvement.
- Purified MAG incubated with PKC confirmed serine phosphorylation.
- Extracellular signals, including neuronal interactions and growth factors, did not alter MAG phosphorylation levels.
Conclusions:
- MAG phosphorylation occurs exclusively on serine residues in both oligodendrocytes and Schwann cells.
- Protein kinase C is involved in the phosphorylation of MAG.
- MAG phosphorylation appears to be regulated by intracellular cues rather than extracellular signals acting on cell surface receptors.