Identification of novel phosphorylation sites required for activation of MAPKAP kinase‐2

MAP kinase‐activated protein (MAPKAP) kinase‐2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST‐MAPKAP kinase‐2 at Thr25 in the proline‐rich N‐terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C‐terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase‐2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P‐labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P‐labelling of Thr338 is likely to result from autophosphorylation. GST‐MAPKAP kinase‐2 lacking the N‐terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full‐length GST‐MAPKAP kinase‐2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N‐terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase‐2, and this constitutively active form may be useful for studying its physiological roles.