K. Engel et al., CONSTITUTIVE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASE-ACTIVATEDPROTEIN-KINASE-2 BY MUTATION OF PHOSPHORYLATION SITES AND AN A-HELIX MOTIF, The Journal of biological chemistry, 270(45), 1995, pp. 27213-27221
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.