Myelin P0 glycoprotein: identification of the site phosphorylated in vitro and in vivo by endogenous protein kinases.
Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO4(3-) from [gamma-32P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181. In the second peptide, Ser197, Ser199, and Ser204 were phosphorylated. All these serines are clustered in the C-terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0. We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197, Ser199, Ser204, Ser208, and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.
Hilmi
S
S
Institut de Biochimie Génétique et Cellulaire du CNRS, Bordeaux, France.
Fournier
M
M
Valeins
H
H
Gandar
J C
JC
Bonnet
J
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D018993:Myelin P0 Protein; D009185:Myelin Proteins / Q000737:chemistry / Q000378:metabolism*; D009186:Myelin Sheath / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012584:Sciatic Nerve / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014018:Tissue Distribution
D018993:Myelin P0 Protein; D009185:Myelin Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases