CONSTITUTIVE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASE-ACTIVATEDPROTEIN-KINASE-2 BY MUTATION OF PHOSPHORYLATION SITES AND AN A-HELIX MOTIF

A recently described downstream target of mitogen-activated protein ki nases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which ha s been shown to be responsible for small heat shock protein phosphoryl ation, We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion prote in, p44(MAPK) and p38/40(MAPK) in vitro and using an epitope-tagged MA PKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carb oxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP k inase 2 in vitro and in vivo is dependent on phosphorylation of a seco nd threonine residue, Thr-205, which is located within the catalytic d omain and which is highly conserved in several protein kinases. Consti tutive activation of MAPKAP kinase 2 is obtained by replacement of bot h of these threonine residues by glutamic acid, A constitutively activ e form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-t erminal region containing Thr-317 and the A-helix motif or by replacin g the conserved residues of the A-helix, These data suggest a dual mec hanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 ins ide the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.