Voltage-sensing Phosphatase Modulation by a C2 Domain

Overview

Journal Front Pharmacol

Date 2015 Apr 24

PMID 25904865

Citations 13

Authors

Paul M Castle

Kevin D Zolman

Susy C Kohout

Affiliations

Soon will be listed here.

Abstract

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in VSP function.

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.

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.

Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltage-sensing phosphatase (VSP).

Mizutani N, Kawanabe A, Jinno Y, Narita H, Yonezawa T, Nakagawa A Proc Natl Acad Sci U S A. 2022; 119(26):e2200364119.

PMID: 35733115 PMC: 9245683. DOI: 10.1073/pnas.2200364119.

The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells.

Ratzan W, Rayaprolu V, Killian S, Bradley R, Kohout S PLoS One. 2019; 14(4):e0209056.

PMID: 30964862 PMC: 6456211. DOI: 10.1371/journal.pone.0209056.

Molecular mechanisms of coupling to voltage sensors in voltage-evoked cellular signals.

Okamura Y, Okochi Y Proc Jpn Acad Ser B Phys Biol Sci. 2019; 95(3):111-135.

PMID: 30853698 PMC: 6541726. DOI: 10.2183/pjab.95.010.

References

Shenoy S, Nanda H, Losche M . Membrane association of the PTEN tumor suppressor: electrostatic interaction with phosphatidylserine-containing bilayers and regulatory role of the C-terminal tail. J Struct Biol. 2012; 180(3):394-408. PMC: 3503488. DOI: 10.1016/j.jsb.2012.10.003. View

Kohout S, Bell S, Liu L, Xu Q, Minor Jr D, Isacoff E . Electrochemical coupling in the voltage-dependent phosphatase Ci-VSP. Nat Chem Biol. 2010; 6(5):369-75. PMC: 2857593. DOI: 10.1038/nchembio.349. View

Kimber W, Trinkle-Mulcahy L, Cheung P, Deak M, Marsden L, Kieloch A . Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo. Biochem J. 2002; 361(Pt 3):525-36. PMC: 1222335. DOI: 10.1042/0264-6021:3610525. View

Aguissa-Toure A, Li G . Genetic alterations of PTEN in human melanoma. Cell Mol Life Sci. 2011; 69(9):1475-91. PMC: 11114653. DOI: 10.1007/s00018-011-0878-0. 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

Hobiger K, Utesch T, Mroginski M, Friedrich T . Coupling of Ci-VSP modules requires a combination of structure and electrostatics within the linker. Biophys J. 2012; 102(6):1313-22. PMC: 3309284. DOI: 10.1016/j.bpj.2012.02.027. View

Lemmon M . Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol. 2008; 9(2):99-111. DOI: 10.1038/nrm2328. View

McCrea H, De Camilli P . Mutations in phosphoinositide metabolizing enzymes and human disease. Physiology (Bethesda). 2009; 24:8-16. PMC: 3499097. DOI: 10.1152/physiol.00035.2008. View

Nalefski E, Falke J . The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci. 1996; 5(12):2375-90. PMC: 2143302. DOI: 10.1002/pro.5560051201. View

10.

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

11.

Simpson L, Parsons R . PTEN: life as a tumor suppressor. Exp Cell Res. 2001; 264(1):29-41. DOI: 10.1006/excr.2000.5130. View

12.

Kurokawa T, Takasuga S, Sakata S, Yamaguchi S, Horie S, Homma K . 3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP). Proc Natl Acad Sci U S A. 2012; 109(25):10089-94. PMC: 3382541. DOI: 10.1073/pnas.1203799109. View

13.

Johnson J, Gray M, Chen C, Mochly-Rosen D . A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J Biol Chem. 1996; 271(40):24962-6. DOI: 10.1074/jbc.271.40.24962. View

14.

Halaszovich C, Schreiber D, Oliver D . Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase. J Biol Chem. 2008; 284(4):2106-13. DOI: 10.1074/jbc.M803543200. View

15.

Corbalan-Garcia S, Sanchez-Carrillo S, Garcia-Garcia J, Gomez-Fernandez J . Characterization of the membrane binding mode of the C2 domain of PKC epsilon. Biochemistry. 2003; 42(40):11661-8. DOI: 10.1021/bi034850d. View

16.

Mutua J, Jinno Y, Sakata S, Okochi Y, Ueno S, Tsutsui H . Functional diversity of voltage-sensing phosphatases in two urodele amphibians. Physiol Rep. 2014; 2(7). PMC: 4187576. DOI: 10.14814/phy2.12061. View

17.

Kohout S, Ulbrich M, Bell S, Isacoff E . Subunit organization and functional transitions in Ci-VSP. Nat Struct Mol Biol. 2007; 15(1):106-8. DOI: 10.1038/nsmb1320. View

18.

Koch M, Holt M . Coupling exo- and endocytosis: an essential role for PIP₂ at the synapse. Biochim Biophys Acta. 2012; 1821(8):1114-32. DOI: 10.1016/j.bbalip.2012.02.008. View

19.

Murata Y, Iwasaki H, Sasaki M, Inaba K, Okamura Y . Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature. 2005; 435(7046):1239-43. DOI: 10.1038/nature03650. View

20.

Nanda H, Heinrich F, Losche M . Membrane association of the PTEN tumor suppressor: neutron scattering and MD simulations reveal the structure of protein-membrane complexes. Methods. 2014; 77-78:136-46. PMC: 4388770. DOI: 10.1016/j.ymeth.2014.10.014. View