Kinetic mechanism and inhibitor characterization of WNK1 kinase.
Kinetic mechanism and inhibitor characterization of WNK1 kinase.
复制标题
WNK1 激酶的动力学机制和抑制剂表征。
作者：
Y. Yagi;K. Abe;K. Ikebukuro;K. Sode
II型假性醛固酮减少症（PHAII）是由两个WNK（含-no- k [Lys]激酶）激酶家族成员突变引起的。我们在这里描述了一种体外WNK1微流控流动性转移实验的发展，用于动力学机制的研究。在微流控芯片上使用毛细管电泳检测既适用于化合物选择，也适用于机理研究，因为该方法具有鲁棒性，而且具有高通量和对ATP浓度不敏感的特点。初始速率与底物浓度的双倒数图显示，WNK的随机顺序活性催化了氧化应激反应激酶1 （OXSR1）的磷酸化。然后从市售文库中的86种激酶中发现WNK1抑制剂。有趣的是，Hck、Lck和Src抑制剂PP1和PP2对WNK1表现出正抑制作用。PP1的抑制模式为纯ATP竞争模式，K(i)值为12.7 microM，对OXSR1肽具有非竞争性抑制作用。从结构上比较，我们发现，由于根据Human Kinome Project的结果，WNK1酶被归类为STEs（酵母不育7、不育11和不育20激酶的同源物），而Hck属于TK（酪氨酸激酶）家族，因此WNK1与PP1相互作用的催化位点残基在Hck中保存良好。我们的结论是，基于化合物的结构定位使我们能够发现激酶之间有趣的关系。这一信息有助于我们筛选不抑制Src、Hck和Lck激酶的特异性WNK1治疗试剂，用于治疗高血压。
Pseudohypoaldosteronism type II (PHAII) is caused by the mutation of two members of the WNK (with-no-K[Lys] kinase) kinase family. We describe here the development of an in vitro WNK1 microfluidic mobility shift assay for kinetic mechanism studies. Assays using capillary electrophoresis on a microfluidic chip are suitable for both compound selection and mechanistic studies, because of the robustness of this method, as well as its high-throughput feature and insensitivity to the ATP concentration. Double-reciprocal plots of the initial rates versus the concentration of the substrate revealed that the random sequential activity of WNK catalyzed OXSR1 (oxidative stress response kinase-1) phosphorylation. WNK1 inhibitors were then found from among 86 kinases in a commercially available library. Interestingly, the Hck, Lck, and Src inhibitors, PP1 and PP2, exhibited positive inhibition against WNK1. The inhibition mode of PP1 was analyzed to be pure ATP competition with a K(i) value of 12.7 microM, showing noncompetitive inhibition against the OXSR1 peptide. From the structure-based comparison, we found that, since the WNK1 enzymes are categorized as STEs (homologues of yeast Sterile 7, Sterile 11, and Sterile 20 kinases) and Hck belongs to the TK (tyrosine kinase) family on the basis of the results of the Human Kinome Project, the residues at the catalytic site of the WNK1 that interact with PP1 were well-conserved in Hck. We concluded that the compound-based structural alignment enabled us to find interesting relationships among the kinases. This information helps us to screen specific WNK1 therapeutic reagents with no inhibition of the Src, Hck, and Lck kinases for the treatment of hypertension.
影响因子：
5.5
作者：
Gagnon, KBE;England, R;Delpire, E
通讯作者：
Delpire, E
DOI：
10.1073/pnas.0604607103
发表时间：
2006-07-18
影响因子：
11.1
作者：
Anselmo, Anthony N.;Earnest, Svetlana;Cobb, Melanie H.
通讯作者：
Cobb, Melanie H.