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.
xxR/KxRxxSxx sequences were phosphorylated with high efficiency by both p70 S6 kinase (p70S6K) and MAPKAP kinase-1. The best substrate for MAPKAP kinase-1 (KKKNRTLSVA) was phosphorylated with a Km of 0.17 microM, and the best substrate for p70S6K (KKRNRTLSVA) with a Km of 1.5 microM. 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 p70S6K) tolerated lack of any residue at n-5 if Arg was present at n-2 and n-3. p70S6K (but not p90S6K) tolerated Thr at position n and absence of any residue at n + 2. The peptide KKRNRTLTV, which combined these features, was relatively selective for p70S6K having a 50-fold higher Vmax/Km than MAPKAP kinase-1. Inactivation of the N-terminal kinase domain of MAPKAP kinase-1, which is 60% identical to p70S6K, abolished activity towards all peptides tested, but the enzyme retained 30-40% of its activity if the C-terminal kinase domain was inactivated.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D013379:Substrate Specificity
D017931:DNA Primers; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa