Myelin P₀ Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract

Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO43− from [γ‐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.

A molecular basis for hereditary motor and sensory neuropathy disorders

“…Treatment of P0-expressing cells with calphostin C, a specific inhibitor of protein kinase C, also inhibits P0-mediated adhesion in a dose-dependent manner, whereas inhibition with pervanadate, an inhibitor of tyrosine phosphorylation, does not. Consistent with these data, PKC has been shown to phosphorylate P0 at these same serine residues in vitro and in vivo [77,78], whereas a termination at amino acid 204 results in a severe demyelinating neuropathy in patients [79]. In addition, we have demonstrated that several isoforms of PKC are dramatically upregulated in sciatic nerves of P0 knockout mice [76].…”

“…Region B contains three serine residues at amino acids 197, 199, and 204 known to be phosphorylated on P0 in vivo [77,78], and we have identified a putative protein kinase C (PKC) binding sequence, RSTK, located between amino acids 198 and 201 [76]. Mutations of each of the serines, at 199 and 204 to alanine residues, inhibit P0-mediated cell-cell adhesion.…”

How Does Protein Zero Assemble Compact Myelin?

“…Known phosphorylation sites are labelled (P). The P-site in brackets is phosphorylated in mice but not conserved in humans [66]. The lines below the alignment denote the individual P0 domains.…”

Tyrosine Phosphorylation of PNS Myelin P0 Occurs in the Cytoplasmic Domain and Is Maximal During Early Development

“…P 0 undergoes extensive phosphorylation, most prominently on serine residues in the cytoplasmic region of the protein (Brunden and Poduslo, 1987 ; Agrawal and Agrawal, 1989 ; Suzuki et al, 1990 ; Hilmi et al, 1995). Recently, we showed that P 0 can also be phosphorylated on tyrosine when rat sciatic nerves are incubated in vitro in the presence of pervanadate, a potent phosphotyrosine phosphatase inhibitor, and obtained initial indications that P 0 tyrosine phosphorylation is greater in nerves from developing as compared with adult rats (Iyer et al, 1996).…”