IDENTIFICATION OF NOVEL PHOSPHORYLATION SITES REQUIRED FOR ACTIVATIONOF 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 phosph orylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-term inal region, Thr222 and Ser272 in the catalytic domain and Thr334 in t he C-terminal domain, Using novel methodology we demonstrate that acti vation of MAPKAP kinase-2 requires the phosphorylation of any two of t he three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser2 72, Thr334 and Thr338 became P-32-labelled in stressed KB cells and la belling was prevented by the specific RK inhibitor SE 203580, establis hing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo, The P-32-labelling of Thr338 is likely to result from autophosphorylat ion. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, b ut activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK, In contrast, full-length GST-MAPKAP kinase-2 was phos phorylated 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 kinas e-2, and this constitutively active form may be useful for studying it s physiological roles.