Role of K364 next to the active site cysteine in voltage-dependent phosphatase activity of Ci-VSP
Role of K364 next to the active site cysteine in voltage-dependent phosphatase activity of Ci-VSP
复制标题
DOI：
10.1016/j.bpj.2023.01.022
复制
发表时间：
2023-06-06
影响因子：
3.4
通讯作者：
Okamura，Yasushi
中科院分区：
文献类型：
--
作者：
Paixao，Ian Costa;Mizutani，Natsuki;Okamura，Yasushi
电压感应磷酸酶（VSP）由与电压门控离子通道相似的电压传感器结构域（VSD）和与10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN）具有显著相似性的细胞质磷酸酶区域组成。膜去极化激活VSD，导致三种磷酸肌醇（磷脂酰肌醇磷酸（PIPs））、PI(3,4,5)P3、PI(4,5)P2和PI(3,4)P2的去磷酸化。与PTEN不同，VSP使pip的3-和5-磷酸去磷酸化，而PTEN表现出严格的3-磷酸特异性。本研究通过生物信息学研究发现，一些哺乳动物的VSP同源物在活性中心基序Cx5R上具有氨基酸多样性，在蛋白酪氨酸磷酸酶和pten相关磷酸酶中具有高度保守性；在袋獾、考拉和草原鹿鼠的VSP同源动物中，与Cx5R基序活性位点半胱氨酸相邻的赖氨酸取代了蛋氨酸，在负鼠中取代了亮氨酸。由于已知PTEN中相应位点的赖氨酸对酶活性至关重要，因此我们试图解决该位点上VSP同源物之间氨基酸多样性的重要性。利用PI(4,5)P2和PI(3,4)P2荧光探针研究了海鞘VSP （Ci-VSP）中K364转化为不同氨基酸，并对其电压依赖性磷酸酶活性进行了研究。所有突变体都保留了5-磷酸酶和3-磷酸酶的活性，这表明与PTEN不同，该位点的赖氨酸对于3-磷酸酶活性是不需要的。值得注意的是，与野生型（WT）相比，K364M突变体的5-磷酸酶和3-磷酸酶活性均有所增加。它也显示出较慢的电压传感器运动动力学。K364M突变体的孔雀石绿测定结果显示，其磷酸酶活性与WT没有显著差异，表明底物结合和电压感应之间的相互作用更紧密。斑马鱼VSP中K364M对应的突变导致PI(4,5)P2的电压依赖性去磷酸化增强。进一步的研究将为了解PIPs磷酸酶的底物偏好以及分子工具的定制提供线索。
Voltage-sensing phosphatase (VSP) consists of the voltage sensor domain (VSD) similar to that of voltage-gated ion channels and the cytoplasmic phosphatase region with remarkable similarity to the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Membrane depolarization activates VSD, leading to dephosphorylation of three species of phosphoinositides (phosphatidylinositol phosphates (PIPs)), PI(3,4,5)P3, PI(4,5)P2, and PI(3,4)P2. VSP dephosphorylates 3- and 5-phosphate of PIPs, unlike PTEN, which shows rigid 3-phosphate specificity. In this study, a bioinformatics search showed that some mammals have VSP orthologs with amino acid diversity in the active center motif, Cx5R, which is highly conserved among protein tyrosine phosphatases and PTEN-related phosphatases; lysine next to the active site cysteine in the Cx5R motif was substituted for methionine in VSP orthologs of Tasmanian devil, koala, and prairie deer mouse, and leucine in opossum. Since lysine at the corresponding site in PTEN is known to be critical for enzyme activities, we attempted to address the significance of amino acid diversity among VSP orthologs at this site. K364 was changed to different amino acids in sea squirt VSP (Ci-VSP), and voltage-dependent phosphatase activity inXenopusoocyte was studied using fluorescent probes for PI(4,5)P2and PI(3,4)P2. All mutants retained both 5-phosphatase and 3-phosphatase activity, indicating that lysine at this site is dispensable for 3-phosphatase activity, unlike PTEN. Notably, K364M mutant showed increased activity both of 5-phosphatase and 3-phosphatase compared with the wild type (WT). It also showed slower kinetics of voltage sensor motion. Malachite green assay of K364M mutant did not show significant difference of phosphatase activity from WT, suggesting tighter interaction between substrate binding and voltage sensing. Mutation corresponding to K364M in the zebrafish VSP led to enhanced voltage-dependent dephosphorylation of PI(4,5)P2. Further studies will provide clues to understanding of substrate preference in PIPs phosphatases as well as to customization of a molecular tool.