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.

Calbindin immunoreactivity of horizontal cells in the developing rabbit retina.

Horizontal cells are retinal interneurons that establish inhibitory feedback loops within the outer plexiform layer of the primary visual pathway. Most mammalian retinas contain two types of horizontal cells. A-type horizontal cells have neuritic branches that contact cone photoreceptors exclusively, while the B-type horizontal cells have dendritic branches that contact cones, in addition to axons that form synapses with rod photoreceptors. Immunoreactivity for calbindin, a calcium binding protein involved in calcium transport, was used as a marker for horizontal cells during post-natal development of the rabbit retina. On post-natal days 1, 3 and 5, calbindin immunoreactivity is limited to a single population of A-type horizontal cells. They appear as a monolayer of cells with broad tapering processes, establishing the proximal border of the nascent outer plexiform layer and forming a target for ingrowing cone photoreceptor terminals. The size and density of the cell bodies and the length of neuritic processes are essentially unchanged during this period, which corresponds to the time of peak expression of GABAergic markers in horizontal cells. Coincident with a decrease in GABAergic markers and the completion of cone-to-horizontal cell synaptogenesis by day 7, changes within the horizontal cell mosaic are detected morphometrically. A delayed phase of overall cell growth results in a 70% increase in average somal diameter (representing a 3.7-fold increase in spherical volume), a six-fold increase in mean neurite length and a decrease in cell density to one-third of that found in the newborn. We conclude that the process of terminal differentiation of horizontal cells is not complete until some time after the second post-natal week. Furthermore, the expression of GABAergic markers is associated primarily with early maturational events, whereas expression of calbindin is sustained throughout post-natal development, suggesting a prominent role for calcium dependent mechanisms at all development stages.

P0 phosphorylation in nerves from normal and diabetic rats: role of protein kinase C and turnover of phosphate groups.

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P0 phosphate groups was studied in normal and experimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [32P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P0, was increased 2-5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P0 were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P0 and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C alpha-isoform. The adenylate cyclase activator, forskolin, had no effect on the PDB-stimulated phosphorylation of P0 in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P0 phosphate groups, nerves were incubated with 32P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P0 from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P0 phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P0 phosphate moieties was accelerated in normal, but not in diabetic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)