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

Kervinen J, Ma H, Bayoumy S, Schubert C, Milligan C, Lewandowski F, Moriarty K, Desjarlais RL, Ramachandren K, Wang H, Harris CA, Grasberger B, Todd M, Springer BA, Deckman I

Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, Pennsylvania 19341, USA. jkervine@prdus.jnj.com

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 4-10mg 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 p38alpha 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, ATPgammaS, 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 microM, 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.

Published 2 May 2006 in Arch Biochem Biophys, 449(1): 47-56.
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