Role of K364 Next to the Active Site Cysteine in Voltage-dependent Phosphatase Activity of Ci-VSP

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 2023 Jan 21

PMID 36680342

Authors

Ian Costa Paixao

Natsuki Mizutani

Makoto Matsuda

Rizki Tsari Andriani

Takafumi Kawai

Atsushi Nakagawa

Yoshifumi Okochi

Yasushi Okamura

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Voltage clamp fluorometry in oocytes to study the voltage-sensing phosphatase.

Young V, Rayaprolu V, Kohout S Bio Protoc. 2025; 15(4):e5212.

PMID: 40028022 PMC: 11865819. DOI: 10.21769/BioProtoc.5212.

The significance of electrical signals in maturing spermatozoa for phosphoinositide regulation through voltage-sensing phosphatase.

Kawai T, Morioka S, Miyata H, Andriani R, Akter S, Toma G Nat Commun. 2024; 15(1):7289.

PMID: 39181879 PMC: 11344830. DOI: 10.1038/s41467-024-51755-2.

Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

Rayaprolu V, Miettinen H, Baker W, Young V, Fisher M, Mueller G J Gen Physiol. 2024; 156(7).

PMID: 38771271 PMC: 11109755. DOI: 10.1085/jgp.202313467.

Membranes in focus.

Sezgin E, Levental I Biophys J. 2023; 122(11):E1-E4.

PMID: 37209687 PMC: 10257145. DOI: 10.1016/j.bpj.2023.05.005.

References

Leitner M, Hobiger K, Mavrantoni A, Feuer A, Oberwinkler J, Oliver D . A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN. Cell Mol Life Sci. 2018; 75(22):4235-4250. PMC: 6182344. DOI: 10.1007/s00018-018-2867-z. View

Lacroix J, Halaszovich C, Schreiber D, Leitner M, Bezanilla F, Oliver D . Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential. J Biol Chem. 2011; 286(20):17945-53. PMC: 3093869. DOI: 10.1074/jbc.M110.201749. View

Hammond G, Burke J . Novel roles of phosphoinositides in signaling, lipid transport, and disease. Curr Opin Cell Biol. 2020; 63:57-67. PMC: 7247936. DOI: 10.1016/j.ceb.2019.12.007. View

Balla T . Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev. 2013; 93(3):1019-137. PMC: 3962547. DOI: 10.1152/physrev.00028.2012. View

Kruse M, Kohout S, Hille B . Reinterpretation of the substrate specificity of the voltage-sensing phosphatase during dimerization. J Gen Physiol. 2019; 151(2):258-263. PMC: 6363406. DOI: 10.1085/jgp.201812260. View

Villalba-Galea C, Sandtner W, Starace D, Bezanilla F . S4-based voltage sensors have three major conformations. Proc Natl Acad Sci U S A. 2008; 105(46):17600-7. PMC: 2584729. DOI: 10.1073/pnas.0807387105. View

Rosasco M, Gordon S, Bajjalieh S . Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase. Biophys J. 2015; 109(12):2480-2491. PMC: 4699894. DOI: 10.1016/j.bpj.2015.11.004. View

Mavrantoni A, Thallmair V, Leitner M, Schreiber D, Oliver D, Halaszovich C . A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases. Front Pharmacol. 2015; 6:68. PMC: 4379879. DOI: 10.3389/fphar.2015.00068. View

Grimm S, Isacoff E . Allosteric substrate switching in a voltage-sensing lipid phosphatase. Nat Chem Biol. 2016; 12(4):261-7. PMC: 4798927. DOI: 10.1038/nchembio.2022. View

10.

Murata Y, Okamura Y . Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. J Physiol. 2007; 583(Pt 3):875-89. PMC: 2277204. DOI: 10.1113/jphysiol.2007.134775. View

11.

Iwasaki H, Murata Y, Kim Y, Hossain M, Worby C, Dixon J . A voltage-sensing phosphatase, Ci-VSP, which shares sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci U S A. 2008; 105(23):7970-5. PMC: 2430346. DOI: 10.1073/pnas.0803936105. View

12.

Kumar S, Stecher G, Li M, Knyaz C, Tamura K . MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018; 35(6):1547-1549. PMC: 5967553. DOI: 10.1093/molbev/msy096. View

13.

Doumane M, Caillaud M, Jaillais Y . Experimental manipulation of phosphoinositide lipids: from cells to organisms. Trends Cell Biol. 2022; 32(5):445-461. DOI: 10.1016/j.tcb.2022.01.009. View

14.

Suh B, Inoue T, Meyer T, Hille B . Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science. 2006; 314(5804):1454-7. PMC: 3579521. DOI: 10.1126/science.1131163. View

15.

Villalba-Galea C, Sandtner W, Dimitrov D, Mutoh H, Knopfel T, Bezanilla F . Charge movement of a voltage-sensitive fluorescent protein. Biophys J. 2009; 96(2):L19-21. PMC: 2716470. DOI: 10.1016/j.bpj.2008.11.003. View

16.

Falkenburger B, Jensen J, Hille B . Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells. J Gen Physiol. 2010; 135(2):99-114. PMC: 2812502. DOI: 10.1085/jgp.200910345. View

17.

Delmas P, Brown D . Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci. 2005; 6(11):850-62. DOI: 10.1038/nrn1785. View

18.

Yen H, Hoi K, Liko I, Hedger G, Horrell M, Song W . PtdIns(4,5)P stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature. 2018; 559(7714):423-427. PMC: 6059376. DOI: 10.1038/s41586-018-0325-6. View

19.

Okamura Y, Kawanabe A, Kawai T . Voltage-Sensing Phosphatases: Biophysics, Physiology, and Molecular Engineering. Physiol Rev. 2018; 98(4):2097-2131. DOI: 10.1152/physrev.00056.2017. View

20.

Okamura Y, Murata Y, Iwasaki H . Voltage-sensing phosphatase: actions and potentials. J Physiol. 2008; 587(3):513-20. PMC: 2670076. DOI: 10.1113/jphysiol.2008.163097. View