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Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity

Authors
  • Kervinen, Jukka
  • Ma, Hongchang
  • Bayoumy, Shariff
  • Schubert, Carsten
  • Milligan, Cynthia
  • Lewandowski, Frank
  • Moriarty, Kevin
  • DesJarlais, Renee L.
  • Ramachandren, Kannan
  • Wang, Haiyun
  • Harris, Crafford A.
  • Grasberger, Bruce
  • Todd, Matthew
  • Springer, Barry A.
  • Deckman, Ingrid
Type
Published Article
Journal
Archives of Biochemistry and Biophysics
Publisher
Elsevier BV
Publication Date
Jan 01, 2006
Volume
449
Issue
1
Pages
47–56
Identifiers
DOI: 10.1016/j.abb.2006.03.018
Source
Elsevier
Keywords
License
Unknown

Abstract

MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4–10 mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1–370, removal of the C-terminal autoinhibitory peptide in 1–338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1–338 by p38α further increased activity 12-fold. A putative constitutively active mutant, 1–370/T222E/T334E, was 6.3-fold less active than phosphorylated 1–370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg 2+-dependent. Residues 1–40 were required for high-affinity binding of ADP, ATPγS, staurosporine, and K252a. A mutation M138A rendered 1–370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 μM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.

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