Comparison of the specificities of p70 S6 kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation

• Published in: FEBS letters Vol. 375; no. 3; pp. 289 - 293
• Main Authors: Leighton, Ian A., Dalby, Kevin N., Barry Caudwell, F., Cohen, Patricia T.W., Cohen, Philip
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20.11.1995
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA Primers; Kinetics; Liver - enzymology; MAP kinase; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides - chemistry; Oligopeptides - metabolism; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Phosphorylation; Point Mutation; Polymerase Chain Reaction; Protein kinase; Protein phosphorylation; Protein-Serine-Threonine Kinases - biosynthesis; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - isolation & purification; Protein-Serine-Threonine Kinases - metabolism; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Ribosomal protein S6; Ribosomal Protein S6 Kinases; Ribosomal Protein S6 Kinases, 90-kDa; S6 kinase; Site-directed mutagenesis; Substrate Specificity; S6 kinase; MAP kinase; Ribosomal protein S6; Protein phosphorylation; Protein kinase; Site-directed mutagenesis
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(95)01170-J

xxR/KxRxxSxx sequences were phosphorylated with high efficiency by both p70 S6 kinase (p70 S6K) and MAPKAP kinase-1. The best substrate for MAPKAP kinase-1 (KKKNRTLSVA) was phosphorylated with a K m of 0.17 μM, and the best substrate for p70 S6K (KKRNRTLSVA) with a K m of 1.5 μM. The requirement of both enzymes for Arg/Lys at position n-5 could be partially replaced by inserting basic residues at other positions, especially by an Arg at n - 2 or n - 4. MAPKAP kinase-1 (but not p70 S6K) tolerated lack of any residue at n - 5 if Arg was present at n - 2 and n - 3. p70 S6K (but not p90 S6K) tolerated Thr at position n and absence of any residue at n + 2. The peptide KKRNRTLTV, which combined these features, was relatively selective for p70 S6K having a 50-fold higher V max/ K m than MAPKAP kinase-1. Inactivation of the N-terminal kinase domain of MAPKAP kinase-1, which is 60% identical to p70 S6K, abolished activity towards all peptides tested, but the enzyme retained 30–40% of its activity if the C-terminal kinase domain was inactivated.