Tyrosine phosphorylation of myelin protein PO.
Po (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 32P in either the presence or absence of phorbol ester. Labeled Po was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial acid hydrolysis. 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 hydroperoxide (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 PO 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 Po. 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-Po and antiphosphotyrosine antibodies coincided on the same immunoblot of myelin proteins and purified Po. These data indicate that one or more tyrosyl residues in Po can be phosphorylated in intact sciatic nerve.
Iyer
S
S
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934, USA.
Rowe-Rendleman
C L
CL
Bianchi
R
R
Eichberg
J
J
D000818:Animals; D006868:Hydrolysis; D008297:Male; D018993:Myelin P0 Protein / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012584:Sciatic Nerve / Q000187:drug effects / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D018993:Myelin P0 Protein; C076715:pervanadate; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014638:Vanadates