Tyrosine phosphorylation of myelin protein P o

P o (M r 30 kDa), the major protein component of peripheral nervous system (PNS) myelin, is known to be phosphorylated by protein kinase C on serine residues at multiple sites. This study was conducted to assess whether other amino acids might be phosphorylated in the protein. Segments of rat sciatic nerve were incubated with 32 P in either the presence or absence of phorbol ester. Labeled P o was isolated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subjected to partial acid hydolysis. Upon separation of the hydrolysis products by either thin‐layer electrophoresis or thin‐layer chromatography, a radioactive spot was detected which comigrated with authentic phosphotyrosine. In other experiments, nerves were incubated with the tyrosine phosphatase inhibitors vanadate or vanadyl hydro‐peroxide (pervanadate). When the nerve homogenate proteins were separated on gels and probed with a monoclonal antibody to phosphotyrosine on Western blots, a positive immune reaction was obtained for a protein species which migrated with the same mobility as P o on Coomassie Blue‐stained gels. In the absence of 2‐mercaptoethanol, this immunoreactive band displayed increased mobility on gels which is characteristic of the migration pattern of P o . The same immunostaining results were obtained using a purified peripheral myelin fraction prepared from nerve homogenates. Furthermore, the positions of immunoreactive bands produced by anti‐P o and anti‐phosphotyrosine antibodies coincided on the same immunoblot of myelin proteins and purified P o . These data indicate that one or more tyrosyl residues in P o can be phosphorylated in intact sciatic nerve. © 1996 Wiley‐Liss, Inc.