Insulin- and phorbol ester-stimulated phosphorylation of ribosomal protein S6.
The relative abilities of insulin and the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) to lead to the phosphorylation of ribosomal protein S6 in vivo were compared in a Reuber H35 hepatoma cell line shown previously to be highly responsive to these agents. In quiescent (serum-starved) cultures of H35 cells incubated with 32Pi, both insulin (10(-7) M) and TPA (1.6 X 10(-6) M) resulted in the marked phosphorylation of S6 compared to the unstimulated cultures as evidenced by an increase in radioactivity associated with S6 and by a corresponding shift in the mobility of phosphorylated S6 during two-dimensional electrophoresis. Following incubation with insulin or TPA, greater than 95% of the phosphate was in derivatives containing four to five phosphate groups. The site-specific phosphorylation of S6 in response to both optimal and suboptimal concentrations of insulin and/or TPA was examined by two-dimensional peptide mapping of the trypsin-digested ribosomal protein S6. The tryptic phosphopeptides of S6 obtained following treatment of the H35 cells with insulin and/or TPA were identical and were the same phosphopeptides as those observed previously following the phosphorylation in vitro of 40 S ribosomal subunits from reticulocytes with purified protease-activated kinase II (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 13998-14002).
Trevillyan
J M
JM
Perisic
O
O
Traugh
J A
JA
Byus
C V
CV
D000818:Animals; D002460:Cell Line; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010704:Phorbols / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
D007328:Insulin; D010704:Phorbols; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D011494:Protein Kinases; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate