Tyrosine phosphorylation of myelin protein Po

Abstract

Po (Mr 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 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 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 anti‐phosphotyrosine 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. © 1996 Wiley‐Liss, Inc.

Src-mediated Tyrosine Phosphorylation of p47 in Hyperoxia-induced Activation of NADPH Oxidase and Generation of Reactive Oxygen Species in Lung Endothelial Cells

“…The hydrolysates were evaporated by N 2 gas, and the residues were taken up in ethanol/water (1:1 v/v). Portions of the acid-hydrolyzed extracts were mixed with authentic mixture of phosphoserine, phosphothreonine, and phosphotyrosine, spotted on Silica Gel 60 plates, and resolved by thin layer chromatography in absolute ethanol, 25% ammonia solution (3.5:2.2 v/v) for 120 min at room temperature; the plates were air-dried, and development was repeated once more in the same solvent, and labeled phosphoamino acids were visualized by autoradiography and standards by spraying the plates with 0.5% ninhydrin in acetone (27).…”

Bone marrow mononuclear cells migrate to the demyelinated sciatic nerve and transdifferentiate into Schwann cells after nerve injury: Attempt at a peripheral nervous system intrinsic repair mechanism

“…Purified myelin was obtained from the proximal and distal regions of crushed nerves as well as from the contralateral sciatic nerves of adult rats by homogenizing the tissue in 2 ml of 0.32 M sucrose containing 1.1 μM leupeptin and 1.02 μM pepstatin A, in a glass‐Teflon homogenizer at 3,000–5,000 rpm. Subcellular fractioning was carried out by a modification of the procedure of Iyer et al (1996). The homogenate was layered on 4 ml of 0.85 M sucrose in 16 × 102 mm tubes and centrifuged for 45 min at 85,000 g in a SW 28 Ti rotor.…”

Proteolipid plasmolipin: localization in polarized cells, regulated expression and lipid raft association in CNS and PNS myelin

“…Sciatic nerves from adult Wistar rats and 15‐day‐old rat pups were dissected and sliced into 0.5‐cm pieces. Segments from four adult nerves were pooled and incubated, essentially according to the procedure of Iyer et al . (1996) in Krebs–Ringer bicarbonate buffer containing 430 μ m freshly prepared vanadyl hydroperoxide (pervanadate), 5 m m sodium pyrophosphate and the protease inhibitors (Hofmann–La Roche, Mannheim, Germany) leupeptin, aprotinin, pepstatin and phenylmethylsulfonyl fluoride at 25 μg/mL, for 1 h at 37°C.…”