Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif.
A recently described downstream target of mitogen-activated protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which has been shown to be responsible for small heat shock protein phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion protein, p44MAPK and p38/40MAPK in vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carboxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second threonine residue, Thr-205, which is located within the catalytic domain and which is highly conserved in several protein kinases. Constitutive activation of MAPKAP kinase 2 is obtained by replacement of both of these threonine residues by glutamic acid. A constitutively active form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-terminal region containing Thr-317 and the A-helix motif or by replacing the conserved residues of the A-helix. These data suggest a dual mechanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 inside the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.
Engel
K
K
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Federal Republic of Germany.
Schultz
H
H
Martin
F
F
Kotlyarov
A
A
Plath
K
K
Hahn
M
M
Heinemann
U
U
Gaestel
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D006358:Hot Temperature; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D000099190:MAP-Kinase-Activated Kinase 2
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D000099190:MAP-Kinase-Activated Kinase 2