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
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, Pennsylvania 19341, USA.
Arch Biochem Biophys. 2006 May 15;449(1-2):47-56. doi: 10.1016/j.abb.2006.03.018. Epub 2006 Mar 31.
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.
丝裂原活化蛋白激酶2（MAPKAPK2）调节肿瘤坏死因子和其他细胞因子的合成，是炎症性疾病的潜在药物靶点。使用杆状病毒感染的昆虫细胞，每升培养基可产生4 - 10毫克的五种蛋白质构建体，并对其激酶活性、热稳定性和配体结合亲和力进行了表征。与构建体1 - 370相比，去除1 - 338中的C末端自抑制肽会导致不稳定但部分活性的非磷酸化酶；p38α对1 - 338的磷酸化进一步使活性增加了12倍。一个假定的组成型活性突变体1 - 370/T222E/T334E的活性比磷酸化的1 - 370低6.3倍。热荧光法（一种平衡配体结合测定法）用于测量各种构建体对核苷酸类似物的亲和力。磷酸化核苷酸的结合是Mg(2+)依赖性的。1 - 40位残基是ADP、ATPγS、星形孢菌素和K252a高亲和力结合所必需的。突变M138A使1 - 370对p38抑制剂SB - 203580和SB - 202190敏感，IC50值分别为17.4和14.1 microM。综上所述，这些研究提供了关于配体与MAPKAPK2结合机制的信息，可用于寻找选择性小分子抑制剂。
