Ci-VSP Is a Depolarization-activated Phosphatidylinositol-4,5-bisphosphate and Phosphatidylinositol-3,4,5-trisphosphate 5′-Phosphatase

Abstract

Phosphoinositides are membrane-delimited regulators of protein function and control many different cellular targets. The differentially phosphorylated isoforms have distinct concentrations in various subcellular membranes, which can change dynamically in response to cellular signaling events. Maintenance and dynamics of phosphoinositide levels involve a complex set of enzymes, among them phospholipases and lipid kinases and phosphatases. Recently, a novel type of phosphoinositide-converting protein (termed Ci-VSP) that contains a voltage sensor domain was isolated. It was already shown that Ci-VSP can alter phosphoinositide levels in a voltage-dependent manner. However, the exact enzymatic reaction catalyzed by Ci-VSP is not known. We used fluorescent phosphoinositidebinding probes and total internal reflection microscopy together with patch-clamp measurements from living cells to delineate substrates and products of Ci-VSP. Upon activation of Ci-VSP by membrane depolarization, membrane association of phosphatidylinositol (PI) (4,5)P 2 -and PI(3,4,5)P 3 -specific binding domains decreased, revealing consumption of these phosphoinositides by Ci-VSP. Depletion of PI(4,5)P 2 was coincident with an increase in membrane PI(4)P. Similarly, PI(3,4)P 2 was generated during depletion of PI(3,4,5)P 3 . These results suggest that Ci-VSP acts as a 5-phosphatase of PI(4,5)P 2 and PI(3,4,5)P 3 . Phosphoinositides (PIs)2 are minor components of all cell membranes and have important and diverse roles in the control of protein function and as organelle-specific membrane labels.Via dynamic concentration changes that result from breakdown or interconversion of PI isoforms, PIs act as bona fide second messengers (reviewed in Ref. 1). PI dynamics can occur in response to a huge variety of cellular events, including the activation of plasma membrane receptors.Knowledge of cellular PI signaling has dramatically increased over the last years. Much of this progress derives from the characterization of a complex set of enzymes that generate, interconvert, and degrade the various PI isoforms (2) and from the introduction of genetically encoded lipid probes that allow the tracking of the dynamics of specific PI isoforms by live cell fluorescence microscopy (3).Further research into two aspects of PI signaling appears particularly promising. First, PI homeostasis and dynamic modulation require the tight spatiotemporal regulation of lipid kinases and phosphatases, which is largely unexplored. The second issue concerns analysis of downstream targets of PI signaling. Because cellular events that change [PI] usually involve other signaling pathways, defining the role of PIs is often not straightforward.Both aspects of PI-related cell biology should benefit from methods for isoform-specific alteration of [PI]. Rapid and reversible manipulation of [PI] is particularly important because effects of long-term alteration of PI levels by overexpression or knockdown of PI-converting enzymes (e.g. Refs. 4 and 5) may be difficult to interpret. Thus, effe...

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

“…phorylate PI(4,5)P 2 upon membrane depolarization (38,39). The time to reach 50% inhibition by CiVSP ( Fig.…”

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P 2 and maintenance of KCNQ2/3 ion channel current

“…Our results are consistent with previous evidence for precursor sources for PM PI(4,5)P 2 synthesis localized to the PM and the Golgi (28, 49, 52-54). For example, following transient activation of voltage-sensitive 5-phosphatase (49,54), the recovery of PM PI(4,5)P 2 from its PM PI(4)P precursor is extremely fast (τ = ∼10 s), suggesting that these pools normally may be in a rapid, dynamic equilibrium. Nevertheless, recent studies monitoring phosphoinositide levels by isotope labeling, mass spectrometry, and antibody staining in fixed cells have advanced the idea that the participation of PM PI(4)P pools in maintaining resting PM PI(4,5)P 2 is truly minor (29)(30)(31).…”

Discovery and functional characterization of a neomorphic PTEN mutation

“…PC-3 cells were purchased from the American Type Culture Collection and maintained in RPMI 1640 with 10% (vol/vol) FBS, and CHO cells were grown as previously described (17). PI3K-p110α inhibitor BYL719 (Selleckchem) and PI3K-p110β inhibitor AZD6428 (Selleckchem) were dissolved in DMSO.…”

“…TIRF imaging was done as described before (17). Briefly, a BX51WI upright microscope (Olympus) equipped with a TIRF-condenser (N.A.…”

“…1 A and B). Curiously, the p.A126G mutation produces a binding pocket amino acid sequence identical to VSP family proteins (14)(15)(16), which are PTEN-homologous phosphoinositide 5-phosphatases (17) (Fig. 1C).…”