TYROSINE PHOSPHORYLATION OF MYELIN PROTEIN P-0

P-0 (M(r) 30 kDa), the major protein component of peripheral nervous s ystem (PNS) myelin, is known to be phosphorylated by protein kinase C on serine residues at multiple sites, This study was conducted to asse ss whether other amino acids might be phosphorylated in the protein, S egments of rat sciatic nerve were incubated with P-32 in either the pr esence or absence of phorbol ester, Labeled P-o was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to p artial acid hydolysis. Upon separation of the hydrolysis products by e ither thin-layer electrophoresis or thin-layer chromatography, a radio active spot was detected which comigrated with authentic phosphotyrosi ne, In other experiments, nerves were incubated with the tyrosine phos phatase inhibitors vanadate or vanadyl hydroperoxide (pervanadate). Wh en the nerve homogenate proteins were separated on gels and probed wit h a monoclonal antibody to phosphotyrosine on Western blots, a positiv e immune reaction was obtained for a protein species which migrated wi th the same mobility as P-0 on Coomassie Blue-stained gels, In the abs ence of 2-mercaptoethanol, this immunoreactive band displayed increase d mobility on gels which is characteristic of the migration pattern of P-0. The same immunostaining results were obtained using a purified p eripheral myelin fraction prepared from nerve homogenates, Furthermore , the positions of immunoreactive bands produced by anti-P-0 and antip hosphotyrosine antibodies coincided on the same immunoblot of myelin p roteins and purified P-0. These data indicate that one or more tyrosyl residues in P-0 can be phosphorylated in intact sciatic nerve. (C) 19 96 Wiley-Liss, Inc.