phosphothreonine and pervanadate

To study the role of protein kinase B (PKB) in response to cellular stress, we examined PKBalpha activity following different stress treatments. Hyperosmotic but not chemical stress resulted in inactivation of PKBalpha and prevented activation by pervanadate and mitogens. Hyperosmotic shock did not affect the MAP kinase pathway, suggesting that this inhibitory effect was specific for PKB. Our data further indicate that downregulation occurs via dephosphorylation of Thr308 and Ser473, the major regulatory phosphorylation sites of PKBalpha. Indeed, calyculin A, which inhibits protein phosphatases 1 and 2A, effectively blocked hyperosmotic stress-mediated inactivation (dephosphorylation) of PKBalpha. High osmolarity did not affect phosphatidylinositol 3-kinase activity but led to a marked increase in PI(3,4,5)P3 and a decrease in PI(3,4)P2 formation after pervanadate stimulation, suggesting that hyperosmotic stress has an inhibitory effect on a phosphatidylinositol 5-phosphatase which converts PI(3,4,5)P3 into PI(3,4)P2. Immunofluorescence studies revealed that membrane translocation, a prerequisite for PKB activation, was not affected by hyperosmotic stress. Our results indicate that hyperosmotic stress can act at two levels: (i) inhibition of phosphorylation of Thr308 and Ser473 by upstream kinases and (ii) by promoting rapid dephosphorylation of these regulatory sites.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; 3T3 Cells; Animals; Biological Transport; Calcium-Calmodulin-Dependent Protein Kinases; COS Cells; Down-Regulation; Humans; Isoenzymes; Membranes; Mice; Mitogens; Osmolar Concentration; Osmotic Pressure; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphoprotein Phosphatases; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Vanadates

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.

Topics: Animals; Hydrolysis; Male; Myelin P0 Protein; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Vanadates