Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

doi:10.1073/pnas.1604218113

Cited by 34 publications

(76 citation statements)

References 34 publications

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“…The results of Anap studies [66] are consistent with the gating loop playing a central role in the regulation of VSP enzyme activity [53]. On the other hand, no large motion was detected in FRET-based measurements using Anap and DPA, which rules out any model that requires a large change in the distance between the membrane and enzyme upon activation of the VSD.…”

Section: Coupling Mechanisms Implicated By Vcf Study Using Fuaamentioning

confidence: 61%

“…Genetic methods using fUAA are versatile tools for detecting structural changes in the cytoplasmic region and a recent application of this method to Ci-VSP [66] has enabled us to gain novel insight into the dynamics of the enzyme region. It is anticipated that more information on the physicochemical basis for the observed alterations in Anap fluorescence within proteins will further expand usefulness of this method.…”

Section: Perspectivesmentioning

confidence: 99%

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Domain-to-domain coupling in voltage-sensing phosphatase

et al. 2017

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Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

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Section: Coupling Mechanisms Implicated By Vcf Study Using Fuaamentioning

confidence: 61%

Section: Perspectivesmentioning

confidence: 99%

Domain-to-domain coupling in voltage-sensing phosphatase

et al. 2017

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“…The latency of the fluorescence change following the voltage step was a few milliseconds (Sakata et al . ). It has been suggested that the C2D plays a key role in keeping the catalytic domain beneath the plasma membrane.…”

Section: Genetic Incorporation Of Anap Revealed the Voltage‐dependentmentioning

confidence: 97%

“…Experiments using Anap demonstrated that upon activation of the VSD, the Ci‐VSP PD changes its conformation to accomplish its catalytic activity (Sakata et al . ). Because PIs, the substrates of Ci‐VSP, are a membrane component, it would make sense for the voltage sensor to regulate enzyme activity by controlling the distance between the PD and the plasma membrane.…”

Section: Fret Analysis Indicates That the Catalytic Domain Stays Benementioning

confidence: 97%

Dynamic structural rearrangements and functional regulation of voltage‐sensing phosphatase

Sakata

Okamura

2018

The Journal of Physiology

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The voltage‐sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region. The latter contains a phosphatase domain and a C2 domain, showing remarkable similarity to the tumour suppressor enzyme PTEN. In VSP, membrane depolarization induces a conformational change in the VSD, which activates the phosphoinositide phosphatase. The final outcome in VSP is enzymatic activity in the cytoplasmic region, unlike in voltage‐gated ion channels where conformational change of the transmembrane pore is induced by the VSD. Therefore, it is crucial to detect structural change in the cytoplasmic catalytic region to gain insights into the operating mechanisms of VSP. This review summarizes a recent study in which a method of genetic incorporation of a non‐canonical amino acid, Anap, was used to detect dynamic membrane voltage‐controlled rearrangements of the structure of the catalytic region of sea squirt VSP (Ci‐VSP). Upon membrane depolarization, both the phosphatase domain and the C2 domain move in a similar time frame, suggesting that the two regions are coupled to each other. Measurement of Förster resonance energy transfer (FRET) between Anap introduced into the C2 domain of Ci‐VSP and dipicrylamine in the cell membrane suggested no large movement of the enzyme towards the membrane. Fluorescence changes in Anap induced by different membrane potentials indicate the presence of multiple conformations of the active enzyme.

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“…An analysis of a voltage sensor mutant of zebrafish VSP with a stabilized intermediate state suggested that there are more than two states of the enzyme with distinct rates, analogous to gear changes in an automobile (16). A recent study in which local structural changes were detected using an unnatural fluorescent amino acid genetically incorporated into the cytoplasmic catalytic region suggested two distinct states of the enzyme with different voltage dependence (17). In such an intermediate state, is the rate of enzymatic activity different from the rate of the fully activated state, but with constant substrate specificity?…”

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Simple scheme of lipid enzyme can explain complex lives of phosphoinositides

Okamura

2016

Proc. Natl. Acad. Sci. U.S.A.

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Phospholipids in biological membranes play multiple roles: They not only allow electrical signaling but also serve as precursors of second messengers in morphogenesis and cancer progression. Voltage-sensing phosphatases (VSPs) (1, 2) are unique membrane proteins that link these two functions. The VSP contains both a cytoplasmic lipid phosphatase and a transmembrane voltage sensor that is similar to the voltage sensor of voltage-gated ion channels. The voltage sensor domain is connected to a downstream cytoplasmic region with remarkable similarity to a tumor suppressor enzyme, phosphatase and tensin homolog (PTEN). PTEN dephosphorylates two species of phosphoinositides (PIs), PI(3,4,5)P 3 and PI(3,4)P 2 , in plasma membranes, antagonizing PI-3 kinase activity. Changes of PTEN's phosphatase activities are known to be associated with many human diseases including cancer, diabetes, and autism spectrum disorders (3). In the VSP, membrane depolarization activates the voltage sensor domain, leading to activation of PI phosphatase activity (4). Despite remarkable structural similarity to PTEN, VSP substrate preference toward PIs differs from substrate preference of PTEN; in particular, VSP dephosphorylates PI(4,5)P 2 , whereas PTEN does not. To understand the physiological roles of VSP, it is important to know the kinetic profiles of the various PIs that can be dephosphorylated by VSP upon membrane potential change. In PNAS, Keum et al. (5) precisely characterize and model the kinetics and voltage dependence of VSP activity toward different PI substrates.Detailed properties of enzymatic activities toward diverse PIs have previously been studied by in vitro enzyme assays using color detection of released phosphate with malachite green or separation of radiolabeled PIs by TLC (6). However, these methods have some limitations because they are difficult to apply to the full-length protein of VSP harboring the transmembrane segments. Several PI-binding motifs, such as the plextrin homology domain, can selectively recognize the headgroup of each species of PI, and fusions of such motifs with fluorescent polypeptides can report dynamic change of each species of PI in living cells. These probes have been used by many laboratories to study multiple types of PIs [PI(3,4,5)P 3 , PI(4,5)P 2 , and PI(3,4)P 2 ] in VSP-expressing cells under different membrane potentials (4, 6-9). One puzzling phenomenon in some of these detailed studies is that upon membrane depolarization, PI(3,4)P 2 initially increases, but it later decreases. These changes depend on the dual-enzyme activity of VSP toward PI(3,4,5)P 3 and PI(3,4)P 2 : PI(3,4,5)P 3 is turned into PI(3,4)P 2 through 5′-phosphatase activity, and PI(3,4)P 2 is then dephosphorylated by 3′-phosphatase activity. Such dynamic regulation of PI(3,4)P 2 by VSP activity might be physiologically relevant, because chick fibroblasts overexpressing VSP form remarkable fine processes that depend on increased PI(3,4)P 2 (10). Interestingly, an increase of PI(3,4)P 2 is observed at a lower voltage th...

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Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Cited by 34 publications

References 34 publications

Domain-to-domain coupling in voltage-sensing phosphatase

Domain-to-domain coupling in voltage-sensing phosphatase

Dynamic structural rearrangements and functional regulation of voltage‐sensing phosphatase

Simple scheme of lipid enzyme can explain complex lives of phosphoinositides

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