Gum R J, McLaughlin M M, Kumar S, Wang Z, Bower M J, Lee J C, Adams J L, Livi G P, Goldsmith E J, Young P R
Department of Molecular Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
J Biol Chem. 1998 Jun 19;273(25):15605-10. doi: 10.1074/jbc.273.25.15605.
Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.
p38丝裂原活化蛋白激酶的吡啶基咪唑抑制剂与ATP竞争结合。ATP口袋中23个残基的突变表明，几个影响吡啶基咪唑光亲和交联剂125I-SB 206718结合的残基并不影响激酶活性，反之亦然，这表明吡啶基咪唑与p38的结合方式不同于ATP。p38的两个紧密同源物SAPK3和SAPK4不受SB 203580抑制，并且在ATP口袋铰链附近与p38有三个氨基酸不同。将p38中的这三个氨基酸替换为SAPK3/4中的氨基酸（第106位苏氨酸、第107位组氨酸和第108位亮氨酸替换为甲硫氨酸、脯氨酸和苯丙氨酸）导致125I-SB 206718交联减少以及对SB 203580的抑制作用丧失。仅将第106位苏氨酸替换为甲硫氨酸导致抑制作用不完全丧失。相反，将p38中的三个氨基酸替换到SAPK3、SAPK4或更远亲的JNK1中会导致被SB 203580抑制，而仅将第106位甲硫氨酸替换为苏氨酸会导致抑制作用减弱。这些结果表明，这三个氨基酸可以赋予SB 203580对至少两类不同的丝裂原活化蛋白激酶的特异性和敏感性。
