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 (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.