Effects of Cyclic AMP and Fluoride on Phosphorylation of Ribosomal Protein S6 and on Protein Synthesis in Rabbit Reticulocytes

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Abstract
The effects of N6,O2-dibutyryl-cAMP (Bt2cAMP) and NaF on the phosphorylation of ribosomal protein S6 and on protein synthesis were examined. Rabbit reticulocytes were incubated in a nutritional medium containing 32Pi in the presence and absence of Bt2cAMP (1 mM) and 3-isobutyl-1-methyl-xanthine (1 mM). In the control cells, 4 phosphorylated derivatives of S6 were observed, with most of the radioactivity in the monophosphorylated form. Upon addition of cyclic nucleotide, a 2-fold increase in the phosphorylation of ribosomal protein S6 was observed. This was accompanied by an increase of radioactive phosphate in the diphosphorylated derivative. No alteration in protein synthesis was observed upon addition of cAMP and analogs of cAMP in conjunction with 3-isobutyl-1-methyl-xanthine or theophylline. The effects of NaF on phosphorylation of S6 and on protein synthesis were also examined. At 5 mM NaF, protein synthesis was inhibited by 85%. A 2.5-fold increase in the phosphorylation of ribosomal protein S6 was observed with an accumulation of 32Pi in the diphosphorylated, triphosphorylated and tetraphosphorylated derivatives. Inhibition of protein synthesis coincided with an increase in the more highly phosphorylated derivatives; an increase of radioactive phosphate in the diphosphorylated derivative could not be correlated with an alteration in globin synthesis.