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

Abstract

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.