Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif

J Biol Chem. 1995 Nov 10;270(45):27213-21. doi: 10.1074/jbc.270.45.27213.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • DNA Primers / genetics
  • Enzyme Activation
  • Hot Temperature
  • In Vitro Techniques
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Phosphorylation
  • Protein Structure, Secondary
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Tumor Cells, Cultured

Substances

  • DNA Primers
  • Intracellular Signaling Peptides and Proteins
  • Recombinant Fusion Proteins
  • MAP-kinase-activated kinase 2
  • Protein-Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases