Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2

EMBO J. 1995 Dec 1;14(23):5920-30. doi: 10.1002/j.1460-2075.1995.tb00280.x.


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 phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated 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 kinase-2, and this constitutively active form may be useful for studying its physiological roles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arsenites / pharmacology
  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Chymotrypsin / metabolism
  • Enzyme Activation
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Mitogen-Activated Protein Kinase 1
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Phosphoserine / metabolism
  • Phosphothreonine / metabolism
  • Protein Kinases*
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Trypsin / metabolism


  • Arsenites
  • Intracellular Signaling Peptides and Proteins
  • Phosphopeptides
  • Recombinant Fusion Proteins
  • Phosphothreonine
  • Phosphoserine
  • Protein Kinases
  • MAP-kinase-activated kinase 2
  • Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinase 1
  • Chymotrypsin
  • Trypsin
  • arsenite