Ribosomal Protein Phosphorylation in Rat Cerebral Cortex

Earlier investigations of ribosomal protein phosphorylation in rat cerebra1 cortex in vitro have been extended to include an analysis of the effects of adenosine 3’:5’-monophosphate (cyclic AMP) on this process. Cerebral cortical slices from ll-day-old rats were incubated for 2 h with [Y”Plorthophosphate, washed free of extracellular radioactivity, then incubated again in the presence of N”,O”-dibutyryl adenosine 3’:5’-monophosphate (dibutyryl cyclic AMP) or related substances. Addition of 1 m.n dibutyryl cyclic AMP consistently stimulated phosphorylation of ribosomal proteins in the cerebra1 40 S subunit, whereas overall phosphorylation of proteins in the 60 S subunit was not increased by the cyclic nucleotide. The minimum dose of dibutyryl cyclic AMP which was effective in stimulating phosphorylation of 40 S ribosomal proteins was about 0.1 mM. Stimulation was also produced with monobutyryl and other dibutyryl derivatives of cyclic AMP, as well as the phosphodiesterase inhibitor, I-methyl-3Gsobutylxanthine. N2,0”-Dibutyryl guanosine 3’:5’-monophosphate and sodium butyrate (1 mM) were without effect on ribosomal protein phosphorylation. Following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, the protein or proteins of the 40 S subunit which exhibited enhanced phosphorylation in the presence of dibutyryl cyclic AMP appeared to be located in a single radioactive band with a mobility which corresponded to a molecular weight of approximately 32,000. Two-dimensional electrophoresis of the ribosomal proteins on polyacrylamide gels containing urea revealed that the small subunit contained three ribosomal proteins which were phosphorylated in cerebra1 cortical tissue in uitro. The most highly labeled protein (S6) had a molecular weight of approximately 32,000 and consisted of at least five distinct species in different states of phosphorylation. Under basal conditions, the bulk of the S6 protein existed in the nonphosphorylated state.