3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P ₂ ] by voltage-sensing phosphatase (VSP)

Abstract

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] and phosphatidylinositol 4,5-bisphosphate [PI (4,5)P 2 ] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P 3 . TLC assay showed that the 3′ phosphate of PI(3,4,5)P 3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] was removed by VSPs. Monitoring of PI(3,4)P 2 levels with the pleckstrin homology (PH) domain from tandem PH domaincontaining protein (TAPP1) fused with GFP (PH TAPP1 -GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P 3 . However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P 3 is dephosphorylated at the 5′ position, PI(3,4)P 2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.phosphoinositide | ascidian P hosphoinositides serve as not only components of biological membranes, but also as coordinators of diverse cellular events including proliferation, cell migration, vesicle turnover, and ion transport. Numerous phosphatases and kinases that regulate phosphoinositide availability have been identified (1, 2), and defects or enhancements of these enzymes lead to tumorigenesis, metabolic disorders, and degeneration. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a wellcharacterized phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (3). Defect or loss of PTEN leads to generation or progression of tumors (4, 5), and enhancement of PTEN underlies diabetes (6). We have shown that a sea squirt ortholog of one PTEN-related phosphatase, transmembrane phosphatase with tensin homology (TPTE)/ TPTE and PTEN homologous inositol lipid phosphatase (TPIP), designated as Ciona intestinalis VSP (Ci-VSP), dephosphorylates phosphoinositides that depend on membrane potential (7, 8). Ci-VSP has a voltage-sensor domain (VSD) consisting of four transmembrane segments and a PTEN-like region. Despite its sequence similarity to PTEN in its active center, Ci-VSP exhibits 5′ phosphatase activity toward PI(3,4,5)P 3 and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], unlike PTEN. Such distinct substrate specificity from PTEN partly d...

A human phospholipid phosphatase activated by a transmembrane control module

“…Moreover, in line with our conclusion, previous functional assays in living cells did not support a role in 3-phosphoinositide signaling ( 11 ), and recent in vitro data are fully consistent with the 5-phosphatase activity ( 25 ). The apparently different enzymatic specifi city previously observed in vitro might therefore point to the importance of the native membrane environment in determining the stereochemistry of interaction of the phosphatase with its phospholipid substrate.…”

“…overexpressed Ci-VSP induced a dissociation of the PI(3,4)P 2 probe TAPP1-PH from the membrane at strongly depolarized potentials ( 25 ). We have not observed such apparent depletion of PI(3,4)P 2 by Ci-VSP in mammalian cells previously ( 2 ).…”

“…In Ci-VSP, this amino acid contributes strongly to the 5-phosphatase specifi city ( 26,27 ). Additional sites, including glutamate 411 in the catalytic gating loop of Ci-VSP, are also important for determining substrate selectivity ( 23,25 ). Further differences in the catalytic center may also contribute to the distinct enzymatic specifi cities; this remains to be addressed.…”

Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

“…Subsequently, VSPs were reported to cleave 3-phosphate from PI(3,4)P 2 , generating PI(4)P upon larger depolarization (20), and, very recently, Ci-VSP was indicated to cleave 3-phosphate from PI(3,4,5)P 3 , generating PI(4,5)P 2 (21,22). Because different substrate reactions are best seen at different voltages, several authors have suggested that changing electric fields can drive VSPs successively through several catalytically active states that favor one set of substrates or reactions over another (20)(21)(22). The active site of VSPs is well conserved among species (12) and shows only a single amino acid difference from the active site of PTEN (1).…”

Depression of voltage-activated Ca2+release in skeletal muscle by activation of a voltage-sensing phosphatase

“…Upon depolarization VSPs dephosphorylate the 5 position of PtdIns(3,4,5)P 3 and PtdIns(4,5)P 2 and the 3 position of PtdIns(3,4)P 2 (Kurokawa et al, 2012). In the present conditions, we tend to favor PtdIns(4,5)P 2 as the active molecule because of its rich history of function in regulating ion channels and transporters in the plasma membrane of various cell types, and also because of our results with the PtdIns(4,5)P 2 -selective PLC 1 PH probe.…”