Abstract
Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of phosphate/mol of protein with both substrates. Peptide map analyses showed that the most prominent phosphorylation sites associated with 40 S substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6. The first serine residue appeared to be the preferred site as has been established previously for 80 S ribosomes (Wettenhall, R.E.H., and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation sites were apparent from the peptide maps. One of these was associated with the triphosphopeptide (termed T1a) having the sequence Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6 (termed Tlc) isolated from mild tryptic digests of whole ribosomes, consisted of the T1a sequence extended by the sequence Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size and chromatographic and isoelectric focusing properties of the T1a/T1c peptides and prominent tryptic peptides of S6 from insulin-stimulated hepatocytes indicated a relationship between these peptides. Thus, it appeared that some of the potential phosphorylation sites in the T1a/T1c region of S6 are phosphorylated by an insulin-regulated kinase in hepatocytes.
Highlights
PRIMARY STRUCTURE OF S6 IN THE REGION OF THE MAJOR PHOSPHORYLATION SITES FOR CAMP-DEPENDENT PROTEIN KINASES*
Low recoveries of the phosphorylated phosphorylation sites associated with 40 S substrates were theadjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6
The accessibility of thethirdand possibly other S6 sites in whole ribosomes to protein kinase might have been impaired by the association between 40 and 60 S ribosomal subunits
Summary
The major phosphoprotein associated with mammalian ribosomes is the 40 S subunit protein termed S6 [1,2,3]. SPtrriumcaturyre of S6 Phosphorylation Sites viously [8] except that the [T-~~PIATPconcentration was increased from 0.2 to 0.3 mM, the overall [32P]phosphate incorporations into the total 80 and 40 S fractions were 5.6 and 3.4 mol of phosphate/mol of ribosomal particles, respectively, during a 60-min incubation In both cases, the amounts of [32P]phosphate incorporatedinto S6 were only about 2mol of phosphate/mol of ribosomal particle as determined from the 32Pradioactivity associated with the S6 band excised from SDS-polyacrylamide gel electrophoretograms (Fig. 1).The additional radioactivity was distributed between several other species only two of which were apparent in two-dimensional electrophoretograms of the ribosomal proteins (Fig. 26’).
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