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Differential phosphorylation of myelin-associated glycoprotein isoforms in cell culture
D E Afar1, J L Salzer, J Roder
1Department of Biochemistry, University of Ottawa, Ontario, Canada.
Abstract:
The alternative splicing of myelin-associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails. Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG. Our results demonstrate that the longer isoform, L-MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues. This phosphorylation is subject to change by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ammonium vanadate, but not by dibutyryl-cyclic AMP. The shorter isoform, S-MAG, is constitutively phosphorylated only on serine residues. While TPA and dibutyryl-cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S-MAG. 32P labeling of v-src-transformed NIH 3T3 cells that express L-MAG also show that L-MAG is likely to be an in vivo substrate for pp60v-src tyrosine kinase activity. These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.
Insights
Myelin-associated glycoprotein (MAG) isoforms show distinct phosphorylation patterns in NIH 3T3 cells. Their in vivo phosphorylation can be modulated by specific compounds, suggesting therapeutic potential.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Alternative splicing of myelin-associated glycoprotein (MAG) mRNA produces two isoforms.
- These isoforms, L-MAG and S-MAG, possess distinct cytoplasmic tail phosphorylation sites.
Purpose of the Study:
- To characterize the in vivo phosphorylation of MAG isoforms in transfected NIH 3T3 cells.
- To investigate the effects of pharmacological agents on MAG isoform phosphorylation.
Main Methods:
- Transfection of NIH 3T3 cells with cDNAs encoding L-MAG and S-MAG.
- In vivo 32P-labeling to detect phosphorylation.
- Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), ammonium vanadate, and dibutyryl-cyclic AMP.
Main Results:
- L-MAG exhibits constitutive serine, threonine, and tyrosine phosphorylation, modulated by TPA and ammonium vanadate.
- S-MAG shows constitutive serine phosphorylation, with ammonium vanadate inducing threonine and tyrosine phosphorylation.
- L-MAG is a potential substrate for pp60v-src tyrosine kinase in transformed cells.
Conclusions:
- Both MAG isoforms are phosphorylated in a heterologous cell system.
- MAG isoform phosphorylation is amenable to pharmacological manipulation.
- Differential phosphorylation of MAG isoforms may influence their function in myelin.