Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity |
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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 |
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Affiliation: | Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, Pennsylvania 19341, USA. jkervine@prdus.jnj.com |
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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 4-10mg 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. |
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