Multisite phosphorylation of a synthetic peptide derived from the carboxyl terminus of the ribosomal protein S6.
The synthetic peptide AKRRRLSSLRASTSKSESSQK (S6-21) which corresponds to the carboxyl-terminal 21 amino acids of human ribosomal protein S6 was synthesized and tested as a substrate for S6/H4 kinase purified from human placenta. The specific activity of the enzyme with the synthetic peptide and 40 S ribosomes was 45 and 23 nmol/min/mg, respectively. The S6/H4 kinase activity with S6-21 was greater than the enzyme activity with any other substrate tested, including histones, protamine, and casein and several other synthetic peptides. The phosphorylation of the peptide was not inhibited by inhibitors of several other proteins kinases. S6/H4 kinase catalyzed the phosphorylation of three major sites in the synthetic peptide and the 40 S ribosomes. A fourth site in S6-21 was phosphorylated more slowly. The principal phosphorylation sites were serines in the acidic carboxyl-terminal domain of the peptide. A serine (Ser-7 or -8) in the amino-terminal domain was phosphorylated at approximately 25% the rate of the carboxyl-terminal domain serines. The data suggest that multiple S6 kinases may be required to phosphorylate S6 at all five sites which are modified in vivo.
Brandon
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SD
Department of Biochemistry, University of North Texas, Denton 76203.
Masaracchia
R A
RA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D038601:Ribosomal Protein S6; D019893:Ribosomal Protein S6 Kinases; D012269:Ribosomal Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity
D010455:Peptides; D010748:Phosphopeptides; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases