Identification of the 40 S ribosomal protein S6 phosphorylation sites induced by cycloheximide

Injection of cycloheximide into rats induced the incorporation of up to 5 mol of phosphate/mol of liver 40 S ribosomal protein S6. Treatment of the protein with cyanogen bromide generated three phosphopeptides of Mr approximately 31,000, approximately 27,000, and approximately 4,000. Increasing the concentration of cyanogen bromide or redigestion of the larger peptides with additional cyanogen bromide converted almost all the phosphate-containing peptides into the Mr approximately 4,000 peptide. This peptide was isolated by reverse phase high pressure liquid chromatography, and the phosphoserines were identified by amino acid sequence analysis after being converted to S-ethyl-cysteine. The results show that the phosphate is incorporated into 5 of 7 serines clustered in a small 13-amino acid segment of the protein. By aligning the sequence of the Mr approximately 4,000 peptide with the recently published mouse cDNA sequence of S6, it is shown that this peptide resides at the carboxyl terminus of the protein. The relevance of the cycloheximide-induced sites of S6 phosphorylation to those stimulated by a variety of mitogens and oncogenes is discussed.