A human phospholipid phosphatase activated by a transmembrane control module
2012; Elsevier BV; Volume: 53; Issue: 11 Linguagem: Inglês
10.1194/jlr.m026021
ISSN1539-7262
AutoresChristian R. Halaszovich, Michael G. Leitner, Angeliki Mavrantoni, Audrey Le, Ludivine Frezza, Anja Feuer, Daniela Schreiber, Carlos A. Villalba‐Galea, Dominik Oliver,
Tópico(s)Ion channel regulation and function
ResumoIn voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P2. In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals. In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P2. In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals. The recent discovery of voltage-sensitive phosphatases (VSPs) (1Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (548) Google Scholar) established a novel molecular principle of electrochemical coupling: VSPs directly mediate the degradation of phosphoinositides in response to depolarization of the membrane potential (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 3Murata Y. Okamura Y. Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2.J. Physiol. 2007; 583: 875-889Crossref PubMed Scopus (132) Google Scholar). These so-far-unique electro-enzymes consist of a transmembrane voltage sensor domain (VSD) homologous to the voltage sensors of voltage-gated ion channels and an intracellular C-terminal catalytic domain (CD) with high similarity to the tumor suppressor lipid phosphatase, PTEN (Fig. 1A) (1Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (548) Google Scholar). The enzymatic activity of the CD is controlled by the VSD via an intramolecular conformational switch (4Kohout S.C. Bell S.C. Liu L. Xu Q. Minor Jr, D.L. Isacoff E.Y. Electrochemical coupling in the voltage-dependent phosphatase Ci-VSP.Nat. Chem. Biol. 2010; 6: 369-375Crossref PubMed Scopus (55) Google Scholar, 5Villalba-Galea C.A. Miceli F. Taglialatela M. Bezanilla F. Coupling between the voltage-sensing and phosphatase domains of Ci-VSP.J. Gen. Physiol. 2009; 134: 5-14Crossref PubMed Scopus (56) Google Scholar). At typical negative resting voltages, the CD is inactive and is rapidly activated at more-positive (depolarized) voltages. The VSP homologs characterized to date are phosphoinositide 5-phosphatases that degrade the major signaling phospholipids PI(4,5)P2 and PI(3,4,5)P3 (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Both phosphoinositides have key signaling roles in many cellular processes, including cell proliferation and differentiation, cytoskeletal dynamics, membrane trafficking, and control of ion channels (6Di Paolo G. De Camilli P. Phosphoinositides in cell regulation and membrane dynamics.Nature. 2006; 443: 651-657Crossref PubMed Scopus (2070) Google Scholar, 7Suh B.C. Hille B. PIP2 is a necessary cofactor for ion channel function: how and why?.Annu. Rev. Biophys. 2008; 37: 175-195Crossref PubMed Scopus (495) Google Scholar). Although little is known to date about the biological functions of VSPs, the ubiquitous roles of phosphoinositides and of electrical signaling suggest a potential impact on a large spectrum of cellular processes. The principle of operation of VSPs was initially demonstrated for Ci-VSP, the prototypic VSP from the invertebrate chordate Ciona intestinalis. Subsequently, functional vertebrate VSPs have been identified in fishes and amphibia (8Hossain M.I. Iwasaki H. Okochi Y. Chahine M. Higashijima S. Nagayama K. Okamura Y. Enzyme domain affects the movement of the voltage sensor in ascidian and Zebrafish voltage-sensing phosphatases.J. Biol. Chem. 2008; 283: 18248-18259Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Ratzan W.J. Evsikov A.V. Okamura Y. Jaffe L.A. Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence.J. Cell. Physiol. 2011; 226: 2740-2746Crossref PubMed Scopus (33) Google Scholar). The VSP gene is also conserved in mammals (10Tapparel C. Reymond A. Girardet C. Guillou L. Lyle R. Lamon C. Hutter P. Antonarakis S.E. The TPTE gene family: cellular expression, subcellular localization and alternative splicing.Gene. 2003; 323: 189-199Crossref PubMed Scopus (68) Google Scholar). In general, there appears to be one VSP homolog in mammalian genomes; in the human genome, however, there are two expressed homologs, TPTE and TPTE2 (also termed TPIP) and additional pseudo-genes (10Tapparel C. Reymond A. Girardet C. Guillou L. Lyle R. Lamon C. Hutter P. Antonarakis S.E. The TPTE gene family: cellular expression, subcellular localization and alternative splicing.Gene. 2003; 323: 189-199Crossref PubMed Scopus (68) Google Scholar, 11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar). TPTE conforms to the architecture of VSPs, but it lacks phosphatase activity due to amino acid exchanges in the catalytic CX5R motif in the P-loop of the phosphatase domain (12Leslie N.R. Yang X. Downes C.P. Weijer C.J. PtdIns(3,4,5)P(3)-dependent and -independent roles for PTEN in the control of cell migration.Curr. Biol. 2007; 17: 115-125Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In contrast, the intracellular domain of TPTE2/TPIP has phosphoinositide phosphatase activity in vitro (11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar). According to its conserved catalytic center, it is apparently the bona-fide ortholog of nonmammalian VSPs and the VSP homologs in other mammals [e.g., mouse PTEN2 (13Wu Y. Dowbenko D. Pisabarro M.T. Dillard-Telm L. Koeppen H. Lasky L.A. PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase.J. Biol. Chem. 2001; 276: 21745-21753Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), also termed mTpte (14Guipponi M. Tapparel C. Jousson O. Scamuffa N. Mas C. Rossier C. Hutter P. Meda P. Lyle R. Reymond A. et al.The murine orthologue of the Golgi-localized TPTE protein provides clues to the evolutionary history of the human TPTE gene family.Hum. Genet. 2001; 109: 569-575Crossref PubMed Scopus (28) Google Scholar)]. For consistent nomenclature, we will thus rename both homologs hVSP1 (for TPIP/TPTE2) and hVSP2 (for human TPTE). Various splice variants of hVSP1 have been reported that mainly differ in the VSD-homologous transmembrane domain. Because all splice variants examined so far in expression systems lacked localization to the plasma membrane, it is unknown whether enzymatic activity is controlled by the VSD-homologous domain and, specifically, whether it is sensitive to membrane potential. Consequently the prevalence of VSP-mediated signaling in mammals has remained unknown. In human and mouse, VSPs are expressed mainly in the testis (10Tapparel C. Reymond A. Girardet C. Guillou L. Lyle R. Lamon C. Hutter P. Antonarakis S.E. The TPTE gene family: cellular expression, subcellular localization and alternative splicing.Gene. 2003; 323: 189-199Crossref PubMed Scopus (68) Google Scholar, 13Wu Y. Dowbenko D. Pisabarro M.T. Dillard-Telm L. Koeppen H. Lasky L.A. PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase.J. Biol. Chem. 2001; 276: 21745-21753Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) but also in brain and stomach (11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar). Cellular expression has been detected in secondary spermatocytes and early spermatids in human and mouse (10Tapparel C. Reymond A. Girardet C. Guillou L. Lyle R. Lamon C. Hutter P. Antonarakis S.E. The TPTE gene family: cellular expression, subcellular localization and alternative splicing.Gene. 2003; 323: 189-199Crossref PubMed Scopus (68) Google Scholar, 13Wu Y. Dowbenko D. Pisabarro M.T. Dillard-Telm L. Koeppen H. Lasky L.A. PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase.J. Biol. Chem. 2001; 276: 21745-21753Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), whereas in the tunicate, Ciona intestinalis, Ci-VSP, is also found in mature sperm (1Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (548) Google Scholar). Thus, a role of VSPs in phosphoinositide signaling in mammalian spermatogenesis or sperm physiology is an exciting possibility. Here, we address the enzymatic properties and the molecular mechanism of activation of hVSP1 using fluorescent sensors for phosphoinositides in living cells. Because hVSP1 localized predominantly to intracellular compartments when transfected into culture cells, we generated a chimeric hVSP1 containing the N-terminus from Ci-VSP that robustly targets to the plasma membrane. Functional assays showed that hVSP1 is a phosphoinositide 5-phosphatase that can be activated via the N-terminal VSD. These findings support a role for VSP-mediated phosphoinositide signaling in mammalian spermatocyte differentiation or sperm function. mRFP-tagged hVSP1CiV was constructed by amplifying the coding sequence for mRFP and amino acids 1 to 239 of Ci-VSP from a pRFP-C1-Ci-VSP plasmid (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A HindIII restriction site was added using reverse primer 5′-ATA TAA GCT TGT TGA TGG GAA TAA AAT ATT C-3′. The resulting amplicon was digested for 30 min at 37°C with fast-digest HindIII, BamHI, and DpnI (Fermentas, St. Leon-Rot, Germany) to avoid carryover of template DNA. Amino acids 120 to STOP of hVSP1-3 [TPIPα (11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar)] were amplified using forward primer 5′-GAC AAG CTT GAA AAG CTG ATG AGA AGG-3′ to insert a guanine to form a HindIII restriction site. The resulting fragment was digested with HindIII and BamHI and ligated to the corresponding mRFP-Ci-VSP fragment. The additional guanine was deleted from the fusion construct by mutagenesis PCR using PfuUltra DNA Polymerase AD (Stratagene, Waldbronn, Germany). The complete coding sequence of the resulting construct was verified by sequencing. Full-length hVSP1 (previously termed TPTE2-1 or TPIPγ, UniProt accession # Q6XPS3) was cloned from a commercial RNA preparation from human testis (Invitrogen; Darmstadt, Germany). Briefly, hVSP1 cDNA was obtained by RT-PCR using semi-nested PCR as described elsewhere (10Tapparel C. Reymond A. Girardet C. Guillou L. Lyle R. Lamon C. Hutter P. Antonarakis S.E. The TPTE gene family: cellular expression, subcellular localization and alternative splicing.Gene. 2003; 323: 189-199Crossref PubMed Scopus (68) Google Scholar). PCR products were subcloned in pJET2.1/blunt vector (Fermentas) and sequenced. Two novel hVSP1 splice variants were identified, lacking either aa 40 to 59 or aa 434 to 464 (numbering according to the full-length sequence). The latter variant was subcloned into pRFP-C, yielding an mRFP-hVSP1 fusion construct. Finally, the C terminus was replaced with the full-length C terminus of hVSP1 isoform 3, yielding the full-length hVSP1 clone. The hVSP1-mRFP fusion construct was obtained by subcloning full-length hVSP1 into pRFP-N vector. The hVSP1hV2-3N chimera was constructed from amino acids 1–50 from hVSP2-3 (TPTEγ, acc. # P56180-3) in pRFP-C vector and amino acids 71–522 from hVSP1. A KpnI restriction site was added at the site of fusion of both constructs using forward mutagenesis primer 5′-GCA TTC AAT GGT ACC ATC CTT TGC ATT TGG-3′. The KpnI restriction site was removed from the fusion construct by mutagenesis PCR. The following plasmids were used: Ci-VSP (UniProt acc. # Q4W8A1) or hVSP variants in pRFP-C or pRFP-N vector; PLCδ1-PH (P51178), Btk-PH (Q06187), OSBP-PH (P22059) in pEGFP-N1 vector; TAPP1-PH (Q9HB21) in FUGW vector (contains eGFP); Bovine phosphatidylinositol 3-kinase p110α (constitutively active mutant K227E; P32871); Lyn11-GFP (P07948) in pcDNA3; and KCNQ4 (Kv7.4, P56696-1) in pBK-CMV. CHO cells plated on glass coverslips or glass-bottom dishes were transfected with these plasmids using JetPEI transfection reagent as described (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Lacroix J. Halaszovich C.R. Schreiber D.N. Leitner M.G. Bezanilla F. Oliver D. Villalba-Galea C.A. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.J. Biol. Chem. 2011; 286: 17945-17953Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). VSPs were transfected individually or cotransfected with either Lyn11-GFP, phosphoinositide sensors, or KCNQ4 potassium channel. For experiments on 3-phosphorylated phosphoinositides, a constitutively active PI-3-kinase [p110α(K227E)] was additionally cotransfected (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Experiments were done 24–48 h posttransfection, with cells selected for robust expression of the VSP constructs on the basis of mRFP fluorescence. CHO cells were whole-cell voltage clamped with an EPC-10 (HEKA; Lambrecht, Germany) or Axopatch 200B amplifier (Molecular Devices; Sunnyvale, CA). Sensing currents were isolated using a P/-10 protocol. Intracellular solution contained (in mM): KCl, 135; MgCl2, 2.5; CaCl2, 2.41; EGTA, 5; HEPES, 5; and Na2ATP, 3, pH 7.3 (with KOH). Extracellular solution, also used for imaging experiments (mM): NaCl, 144; KCl, 5.8; NaH2PO4, 0.7; glucose, 5.6; CaCl2, 1.3; MgCl2, 0.9; and HEPES, 10; pH 7.4 (with NaOH). Patch pipettes were pulled from borosilicate glass (Science Products; Hofheim, Germany). KCNQ4 (Kv7.4) current recordings were sampled at 5 kHz and low-pass-filtered at 2 kHz. Series resistance in these experiments typically was below 5 MΩ and was compensated by 80–90%. Subcellular localization was imaged in living cells with a Zeiss LSM710 confocal microscope (Carl Zeiss AG; Jena, Germany) equipped with a W-Plan Apochromat 20×/1.0 DIC M27 objective. Laser lines used were 561 nm for mRFP and 488 nm for GFP, and detection wavelength ranges were 582–754 nm and 493–582 nm, respectively. TIRF imaging was done as described previously (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, a BX51WI upright microscope equipped with a TIRF-condenser (Olympus) and a 488 nm laser was used. Images were acquired with a CCD camera using TILLvision software (TILL-photonics; Gräfelfing, Germany). Where applicable, the electrophysiology setup was synchronized to the imaging setup. The frame interval was 3 s. TIRF imaging data were analyzed using TILLvision and IgorPro (Wavemetrics; Lake Oswego, OR). Regions of interest (ROIs) encompassed the footprint of a single cell, excluding cell margins to avoid movement artifacts. Normalized fluorescence intensity (F/F0) was calculated pixelwise from the TIRF signal intensity F and the initial fluorescence intensity F0, which was calculated as the average over the baseline interval. Background correction was applied in this process. The F/F0 trace was then calculated by framewise averaging over the ROI. F/F0 traces were corrected for bleaching according to monoexponential fits to the baseline interval as described before (15Lacroix J. Halaszovich C.R. Schreiber D.N. Leitner M.G. Bezanilla F. Oliver D. Villalba-Galea C.A. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.J. Biol. Chem. 2011; 286: 17945-17953Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Steady-state fluorescence voltage relations were derived as reported previously (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Data obtained from individual cells were fitted with a Boltzmann function F = Fmin+ (Fmax-Fmin)/[1 + exp((V-V1/2)/s))], where F is the recorded TIRF intensity, V is the holding potential, V1/2 is the voltage at the half-maximal response, and s describes the steepness of the curve. Data were normalized to Fmax-Fmin as derived from the fit for each individual cell. For expression in Xenopus oocytes, hVSPCiV was cloned into the pBSTA vector. Point mutations were made by PCR using mismatched primers containing the mutation. For RNA synthesis, DNA was linearized with NotI and transcribed using T7 RNA polymerase. KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) constructs for expression in Xenopus oocytes (vector pTLN; kindly provided by T. Jentsch) were linearized with MluI and HpaI, respectively, and RNA was transcribed using SP6 RNA polymerase (Ambion, Frankfurt, Germany). For coexpression of KCNQ channels with potassium currents, Xenopus oocytes were injected with 10 ng of RNA of each construct and incubated for 2–3 days at 12–18°C in a solution containing 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES, pH 7.5. For sensing currents, Xenopus oocytes were injected with 20–50 ng of RNA, and electrophysiological measurements were performed after 3 days of incubation at 15°C. Potassium currents mediated by KCNQ channels were recorded by two-electrode voltage clamp. The extracellular recording solution contained 4–8 mM KOH, 112–116 mM NMG, 120 mM MeSO3 (methanesulfonate), 10 mM HEPES, and 2 mM CaCl2, pH 7.4. Sensing currents were measured 3–4 days after injection with the cut-open oocyte voltage clamp technique as described previously (16Villalba-Galea C.A. Sandtner W. Starace D.M. Bezanilla F. S4-based voltage sensors have three major conformations.Proc. Natl. Acad. Sci. USA. 2008; 105: 17600-17607Crossref PubMed Scopus (167) Google Scholar). The external recording solutions contained 120 mM NMG-MeSO3 (methanesulfonate), 10 mM HEPES, and 2 mM CaCl2, pH 7.4, whereas internal solutions contained 120 mM NMG-MeSO3, 10 mM HEPES, and 2 mM EGTA, pH 7.4. Currents were measured in response to voltage steps (10 s interval) from a holding potential of −60 mV, without leak subtraction during acquisition. Capacitance transient currents were compensated using the amplifier's compensation circuit. Recordings were performed using a homemade acquisition software based on the graphical programming environment LabVIEW (National Instruments; Austin, TX). The software controlled an NI USB-6251 BNC interface (National Instruments). All experiments were performed at room temperature. Data are given as mean ± standard error of the mean (± SE). hVSP1 may be subject to extensive splicing yielding differentially truncated VSD domains (10Tapparel C. Reymond A. Girardet C. Guillou L. Lyle R. Lamon C. Hutter P. Antonarakis S.E. The TPTE gene family: cellular expression, subcellular localization and alternative splicing.Gene. 2003; 323: 189-199Crossref PubMed Scopus (68) Google Scholar). The described N-terminal truncations appeared incompatible with the function of the domain as a voltage sensor. Moreover, truncated splice variants were not targeted to the plasma membrane (see supplementary Fig. I) (11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar). We therefore cloned the full-length variant (hVSP1-1) that corresponds to the canonical isoform according to UniProt (accession # Q6XPS3) (17Jain E. Bairoch A. Duvaud S. Phan I. Redaschi N. Suzek B.E. Martin M.J. McGarvey P. Gasteiger E. Infrastructure for the life sciences: design and implementation of the UniProt website.BMC Bioinformatics. 2009; 10: 136Crossref PubMed Scopus (407) Google Scholar). When transfected into CHO cells (Fig. 1B) or HEK293 cells (see supplementary Fig. IB), localization of even this full-length protein was restricted to intracellular compartments, possibly the Golgi apparatus, as suggested previously for mouse VSP1 (mPTEN2) (13Wu Y. Dowbenko D. Pisabarro M.T. Dillard-Telm L. Koeppen H. Lasky L.A. PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase.J. Biol. Chem. 2001; 276: 21745-21753Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This subcellular localization was not the result of N-terminal mRFP fusion, because hVSP1-1 with a C-terminal mRFP showed the same localization (see supplementary Fig. IB). Accordingly, electrophysiological examination did not reveal sensing currents, the electrical signature of a functional VSD as found in Ci-VSP (Fig. 1C) (1Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (548) Google Scholar). Moreover, the intracellular localization of hVSP1 in the expression system prohibited a direct assessment of voltage-dependent activity of this putative lipid phosphatase. Interestingly, a short splice variant of the paralog, hVSP2-3 (TPTEγ), targets robustly to the plasma membrane (11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar), although functional examination showed absence of sensing currents, indicating a nonfunctional VSD in hVSP2 (see supplementary Fig. IC). Given the distinct subcellular localization of both hVSPs, we considered a possible role of their distinct cytoplasmic N-termini in membrane targeting. However, replacement of the hVSP1 N-terminus with the corresponding terminus of hVSP2-3 also failed to localize the protein to the plasma membrane (see supplementary Fig. IA, B). We therefore adopted an approach that we developed previously to examine the function of phosphoinositide phosphatases (15Lacroix J. Halaszovich C.R. Schreiber D.N. Leitner M.G. Bezanilla F. Oliver D. Villalba-Galea C.A. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.J. Biol. Chem. 2011; 286: 17945-17953Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Thus, we generated a chimeric hVSP1 variant by replacing its entire VSD with the highly homologous domain of the prototypic Ci-VSP (Fig. 1A, lower panel). We will refer to this chimera as hVSP1CiV. hVSP1CiV was robustly targeted to the plasma membrane (Fig. 1B). Depolarizing voltage steps revealed sensing currents similar to Ci-VSP, indicating intact functionality of the VSD in the chimeric hVSP1 (Fig. 1C). Expression of hVSP1CiV in Xenopus oocytes confirmed functionality and allowed for a detailed examination of sensing currents, revealing the sigmoidal dependence on membrane voltage that characterizes voltage sensor domains (see supplementary Fig. II). We tested for lipid phosphatase activity of hVSP1CiV in vivo, using GFP-fused phosphoinositide binding protein domains as fluorescent phosphoinositide sensors (18Varnai P. Balla T. Live cell imaging of phosphoinositide dynamics with fluorescent protein domains.Biochim. Biophys. Acta. 2006; 1761: 957-967Crossref PubMed Scopus (120) Google Scholar). Association of these probes to the membrane reports on the concentration of specific phosphoinositide species, and was measured quantitatively by TIRF microscopy (2Halaszovich C.R. Schreiber D.N. Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase.J. Biol. Chem. 2009; 284: 2106-2113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Lacroix J. Halaszovich C.R. Schreiber D.N. Leitner M.G. Bezanilla F. Oliver D. Villalba-Galea C.A. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.J. Biol. Chem. 2011; 286: 17945-17953Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In cells coexpressing hVSP1CiV and the PI(4,5)P2 sensor PHPLCδ1-GFP, depolarization of the membrane potential via whole-cell patch clamping resulted in the reversible dissociation of the probe from the membrane (Fig. 2A, B) indicating depletion of PI(4,5)P2. This depletion resulted from phosphatase activity of hVSP1, because effects on PHPLCδ1 localization and PI(4,5)P2 concentration were abolished when an inactivating mutation (C363S) was introduced into the catalytic CX5R motif (Fig. 2B; cf. Refs. 15Lacroix J. Halaszovich C.R. Schreiber D.N. Leitner M.G. Bezanilla F. Oliver D. Villalba-Galea C.A. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.J. Biol. Chem. 2011; 286: 17945-17953Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19Maehama T. Taylor G.S. Dixon J.E. PTEN and myotubularin: novel phosphoinositide phosphatases.Annu. Rev. Biochem. 2001; 70: 247-279Crossref PubMed Scopus (407) Google Scholar). Thus, hVSP1 has phosphoinositide phosphatase activity in vivo, and, specifically, dephosphorylates PI(4,5)P2. Moreover, the depletion of PI(4,5)P2 in response to depolarization shows that in hVSP1CiV, enzymatic activity is regulated by membrane voltage via the attached VSD. Initial in vitro work on the isolated CD suggested that hVSP1 has 3-phosphatase activity (11Walker S.M. Downes C.P. Leslie N.R. TPIP: a novel phosphoinositide 3-phosphatase.Biochem. J. 2001; 360: 277-283Crossref PubMed Scopus (119) Google Scholar), which would be incompatible with the observed consumption of cellular PI(4,5)P2 but should instead produce PI(4,5)P2 from PI(3,4,5)P3 (15Lacroix J. Halaszovich C.R. Schreiber D.N. Leitner M.G. Bezanilla F. Oliver D. Villalba-Galea C.A. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.J. Biol. Chem. 2011; 286: 17945-17953Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We therefore performed a detailed analysis of the enzymatic specificity of hVSP1, using fluorescent probes with distinct phosphoinositide specificities. Activation of hVSP1CiV by depolarization resulted in an increase of the PI(4)P concentration, as shown by translocation of PHOSBP-GFP from the cytosol to the plasma membrane (Fig. 2C). Quantitative considerations indicate that the additional PI(4)P results from dephosphorylation of PI(4,5)P2. Thus, PI(4,5)P2 and PI(4)P together make up the bulk of phosphoinositides in the plasma membrane, whereas all other phosphoinositides have much lower steady-state concentrations in this compartment (6Di Paolo G. De Camilli P. Phosphoinositides in cell regulation and membrane dynamics.Nature. 2006; 443: 651-657Crossref PubMed Scopus (2070) Google Scholar, 20Falkenburger B.H. Jensen J.B. Hille B. Kinetics of PIP2
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