An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation
An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation
复制标题
DOI：
10.1016/j.abb.2007.04.004
复制
发表时间：
2007-08-01
影响因子：
3.9
通讯作者：
May, Earl W.
中科院分区：
文献类型：
--
作者：
Rominger, Cynthia M.;Schaber, Michael D.;May, Earl W.
我们已经开发了一种高灵敏度的MEK介导的ATP水解的测定方法，该方法通过丙酮酸激酶和乳酸脱氢酶将ADP的形成与NADH氧化偶联。在不存在蛋白质底物ERK的情况下，磷酸化的NIEK催化稳健的ATP水解。这种ERK-解偶联的ATP酶活性依赖于NIEK的磷酸化状态，并被选择性NIEK激酶抑制剂U 0126消除。ADP的产生伴随着Raf-mediated的MEK磷酸化。基于这一发现，开发了用于测量Raf活性的Raf/MEK偶联测定。根据稳态假设推导的动力学处理，提出了用于分析由该耦合测定产生的反应进度曲线。我们已经表明，选择性Raf抑制剂的抑制效力可以通过该测定准确地确定。(c)2007年爱思唯尔公司All rights reserved.
We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated NIEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of NIEK and is abrogated by the selective NIEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay. (c) 2007 Elsevier Inc. All rights reserved.