Identification of a novel inhibitor of mitogen-activated protein kinase kinase
Identification of a novel inhibitor of mitogen-activated protein kinase kinase
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DOI：
10.1074/jbc.273.29.18623
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发表时间：
1998-07-17
影响因子：
4.8
通讯作者：
Trzaskos, JM
中科院分区：
文献类型：
--
作者：
Favata, MF;Horiuchi, KY;Trzaskos, JM
在基于细胞的报告实验中，化合物U0126(1，4-diamino-2，3-dicyano-1，4-bis[2-aminophenylthio]butadiene)被鉴定为AP-1反式激活的抑制剂。U0126还能抑制含有AP-1反应元件的内源性启动子，但不影响其启动子中缺少AP-1反应元件的基因。U0126的这些作用是由于直接抑制丝裂原激活的蛋白激酶家族成员MEK-1和MEK-2所致。对MEK-1和-2的抑制是选择性的，因为U0126对蛋白激酶C、Ab1、Raf、Mekk、ERK、JNK、MKK-3、MKK-4/SEK、MKK6、CDK2或CDK4的活性几乎没有影响。U0126和MEK抑制剂PD098059(Dudley，D.T.，Pang，L.，Decker，S.J.，Bridges，A.J.和Saltiel，A.R.(1995)Proc.娜塔莉。阿卡德。SCI U.S.A.92,7686-7689)证明了U0126和PD098059对于两种MEK底物ATP和ERK都是非竞争性抑制剂。我们进一步证明了这两个化合物以互斥的方式与S218E/S222D MEK结合，表明它们可能共享一个共同或重叠的结合部位(S)。定量评估这些化合物对MEK抑制的稳态动力学表明，U0126对Delta N3-S218E/S222D MEK的亲和力比PD098059高约100倍。我们进一步测试了这些化合物对激活后从刺激细胞中分离的野生型MEK活性的影响。令人惊讶的是，与Delta N3-S218E/S222D突变酶相比，我们观察到这两个化合物对野生型MEK的亲和力显著降低。这些结果表明，这两种化合物的亲和力是由两种激活的MEK形式之间的微小构象差异所介导的。U0126对MEK的亲和力，对MEK的选择性，以及它的细胞效能表明，该化合物将成为体外和细胞研究丝裂原活化蛋白激酶介导的信号转导的有力工具。
The compound U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for Delta N3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the Delta N3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.