Tumor-promoting phorbol esters stimulate the phosphorylation of ribosomal protein S6 in quiescent Reuber H35 hepatoma cells.
The addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) to serum-starved quiescent Reuber H35 hepatoma cells results in a rapid 5- to 11-fold increase in the incorporation of 32Pi into a Mr = 32,000 ribosomal protein. The Mr = 32,000 protein was the major phosphorylated protein extracted from isolated 80 S ribosomes and was identified as the 40 S ribosomal protein S6 based upon its migration in two-dimensional gels. Insulin, which has been demonstrated to increase the phosphorylation of S6 in a number of cell lines, caused a 10- to 20-fold increase in the incorporation of 32Pi into this Mr = 32,000 ribosomal protein. S6 phosphorylation was dose- and time-dependent being detected as early as 5 min following the addition of 1.6 microM TPA. Maximal phosphorylation of ribosomal protein S6 was achieved by 60 min and remained elevated for at least 90 min in the presence of TPA. The 50% effective dose for TPA was estimated to be 0.14 microM. Based upon the altered migration of S6 in pH 8.5 urea-polyacrylamide gels, it was demonstrated that the increased 32Pi labeling of S6 by TPA was due to a net increase in the incorporation of phosphates into the S6 molecule. Non-tumor-promoting phorbol esters were ineffective in increasing the phosphorylation of S6. In whole cells, exogenously added 1 mM 8-bromoadenosine 3':5'-monophosphate failed to substantially increase phosphorylation of S6 suggesting that the TPA-induced phosphorylation of S6 occurs via a cyclic AMP-independent mechanism. The S6 amino acid residue phosphorylated in response to TPA was phosphoserine. A possible role for protein kinase C in the phosphorylation of ribosomal protein S6 is discussed.
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010704:Phorbols / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors
D010703:Phorbol Esters; D010704:Phorbols; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate