title: Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity.
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.
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, Pennsylvania 19341, USA. jkervine@prdus.jnj.com
authors.forename: Jukka
authors.initials: J
authors.lastname: Kervinen
authors.forename: Hongchang
authors.initials: H
authors.lastname: Ma
authors.forename: Shariff
authors.initials: S
authors.lastname: Bayoumy
authors.forename: Carsten
authors.initials: C
authors.lastname: Schubert
authors.forename: Cynthia
authors.initials: C
authors.lastname: Milligan
authors.forename: Frank
authors.initials: F
authors.lastname: Lewandowski
authors.forename: Kevin
authors.initials: K
authors.lastname: Moriarty
authors.forename: Renee L
authors.initials: RL
authors.lastname: Desjarlais
authors.forename: Kannan
authors.initials: K
authors.lastname: Ramachandren
authors.forename: Haiyun
authors.initials: H
authors.lastname: Wang
authors.forename: Crafford A
authors.initials: CA
authors.lastname: Harris
authors.forename: Bruce
authors.initials: B
authors.lastname: Grasberger
authors.forename: Matthew
authors.initials: M
authors.lastname: Todd
authors.forename: Barry A
authors.initials: BA
authors.lastname: Springer
authors.forename: Ingrid
authors.initials: I
authors.lastname: Deckman
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry; D008024:Ligands; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008024:Ligands; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D000099190:MAP-Kinase-Activated Kinase 2