Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum.
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.
Bandi
H R
HR
Friedrich Miescher Institute, Basel, Switzerland.
Ferrari
S
S
Krieg
J
J
Meyer
H E
HE
Thomas
G
G
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001769:Blood; D003488:Cyanogen Bromide; D008666:Metalloendopeptidases; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D012694:Serine; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide