Department of Physiology, Graduate School of Medicine, Osaka University, Suita, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
Department of Physiology, Graduate School of Medicine, Osaka University, Suita, Japan.
Biophys J. 2023 Jun 6;122(11):2267-2284. doi: 10.1016/j.bpj.2023.01.022. Epub 2023 Jan 20.
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)P, PI(4,5)P, and PI(3,4)P. 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, CxR, which is highly conserved among protein tyrosine phosphatases and PTEN-related phosphatases; lysine next to the active site cysteine in the CxR 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 in Xenopus oocyte was studied using fluorescent probes for PI(4,5)P and PI(3,4)P. 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)P. Further studies will provide clues to understanding of substrate preference in PIPs phosphatases as well as to customization of a molecular tool.
电压感应磷酸酶（VSP）由类似于电压门控离子通道的电压传感器结构域（VSD）和细胞质磷酸酶结构域组成，其与染色体 10 上缺失的磷酸酶和张力蛋白同源物（PTEN）具有显著的相似性。膜去极化激活 VSD，导致三种磷酸肌醇（PI）磷酸酯（PIPs）、PI（3,4,5）P、PI（4,5）P 和 PI（3,4）P 的去磷酸化。与 PTEN 不同，VSP 显示出刚性的 3-磷酸特异性，其可去磷酸化 PIPs 的 3-和 5-磷酸。本研究通过生物信息学搜索发现，一些哺乳动物具有 VSP 直系同源物，其在活性中心基序 CxR 中具有氨基酸多样性，该基序在蛋白酪氨酸磷酸酶和 PTEN 相关磷酸酶中高度保守；CxR 基序中活性位点半胱氨酸旁边的赖氨酸被塔斯马尼亚恶魔、树袋熊和草原鹿鼠的 VSP 直系同源物中的蛋氨酸取代，而负鼠中的亮氨酸取代。由于已知 PTEN 中相应位点的赖氨酸对于酶活性至关重要，因此我们试图解决该位点 VSP 直系同源物中氨基酸多样性的意义。用海鞘 VSP（Ci-VSP）中的不同氨基酸替换 K364，并使用 PI（4,5）P 和 PI（3,4）P 的荧光探针研究 Xenopus oocyte 中的电压依赖性磷酸酶活性。所有突变体均保留了 5-磷酸酶和 3-磷酸酶活性，表明该位点的赖氨酸对于 3-磷酸酶活性不是必需的，与 PTEN 不同。值得注意的是，与野生型（WT）相比，K364M 突变体的 5-磷酸酶和 3-磷酸酶活性均增加。它还显示出电压传感器运动的较慢动力学。与 WT 相比，Malachite green 测定法未显示 K364M 突变体的磷酸酶活性有显著差异，表明底物结合与电压感应之间的相互作用更紧密。在斑马鱼 VSP 中的对应于 K364M 的突变导致 PI（4,5）P 的电压依赖性去磷酸化增强。进一步的研究将为理解 PIPs 磷酸酶中的底物偏好以及定制分子工具提供线索。
