Xu Bing-e, Min Xiaoshan, Stippec Steve, Lee Byung-Hoon, Goldsmith Elizabeth J, Cobb Melanie H
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas 75390-9041, USA.
J Biol Chem. 2002 Dec 13;277(50):48456-62. doi: 10.1074/jbc.M207917200. Epub 2002 Oct 8.
WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.
WNK家族蛋白激酶是一种大型酶，与所有其他蛋白激酶相比，其催化赖氨酸处于独特位置。这些酶与一种基因定义的高血压形式有关。在本研究中，我们引入突变以检验关于催化赖氨酸位置的假设，并研究了WNK1活性调节所涉及的机制。通过对酶片段的分析和序列比对，我们鉴定出了WNK1的一个自抑制结构域。这个分离的结构域在所有四种WNK中都保守，抑制了WNK1激酶结构域的活性。该自抑制结构域中两个关键残基的突变减弱了其抑制WNK激酶活性的能力。与这些结果一致，包含自抑制结构域的WNK1片段中的相同突变增加了其激酶活性。我们还发现，在细菌中表达的WNK1会发生自磷酸化；其活性需要在激活环中的丝氨酸382上进行自磷酸化。
