Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum

Abstract

All of the phosphorylation sites in 40 S ribosomal protein S6 derived from serum-stimulated Swiss mouse 3T3 cells are found within a small cyanogen bromide (CNBr) peptide derived from the carboxyl terminus, Lys218-Lys249. Further cleavage of the CNBr peptide or the intact protein with endoproteinase Lys-C (endo Lys-C) generated a single phosphorylated peptide, implying that all the sites of phosphorylation resided either between Arg231 and Lys243 or between Arg231 and Lys249 if cleavage at Lys243 was blocked by phosphorylation at a nearby residue. To discern between these possibilities and to identify the phosphorylation sites, the protein was purified from serum-stimulated cells and cleaved with endo Lys-C, and the single endo Lys-C phosphorylated peptide was isolated and sequenced following conversion of all the phosphorylated serines to S-ethylcysteine. The results show that the phosphorylated peptide extends from Arg231 to Lys249 and that the sites of phosphorylation in vivo are Ser235, Ser240, Ser244, and Ser247.