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

FEBS Lett. 1995 Nov 20;375(3):289-93. doi: 10.1016/0014-5793(95)01170-j.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • Kinetics
  • Liver / enzymology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Point Mutation
  • Polymerase Chain Reaction
  • 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 Kinases
  • Ribosomal Protein S6 Kinases, 90-kDa
  • Substrate Specificity

Substances

  • DNA Primers
  • Oligopeptides
  • Peptide Fragments
  • Recombinant Proteins
  • Protein Serine-Threonine Kinases
  • Ribosomal Protein S6 Kinases
  • Ribosomal Protein S6 Kinases, 90-kDa