Crystal Structure of the Cytoplasmic Phosphatase and Tensin Homolog (PTEN)-like Region of Ciona intestinalis Voltage-sensing Phosphatase Provides Insight into Substrate Specificity and Redox Regulation of the Phosphoinositide Phosphatase Activity

Abstract

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wildtype (248 -576), wild-type (236 -576), and G365A mutant (248 -576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.The voltage-sensing phosphatase (VSP) 3 was discovered through the survey of the genome of the ascidian, Ciona intestinalis, as a hybrid protein that has a voltage sensor domain (VSD) consisting of four ␣-helices (S1-S4) for voltage sensing and a cytoplasmic region encoding a phosphatidylinositol phosphatase domain (1). The VSP gene is conserved from sea urchins to humans where it is expressed in the testis (2). In the recent report, C. intestinalis VSP (Ci-VSP) is expressed in the cells of the stomach, intestine, and blood of juveniles detected by whole mount in situ hybridization (3). The VSD of VSP bears homology to the VSD of voltage-gated ion channels and a recently identified voltage-gated proton channel protein that contains only the VSD without pore domain, VSOP or Hv1 (4, 5). The Ci-VSP cytoplasmic region, which consists of a phosphatase domain (PD) and a C2 domain, shares high sequence similarity with the phosphatase and tensin homolog (PTEN) (1, 6). The amino acid sequence of the Ci-VSP cytoplasmic region has similarity to that of PTEN with 36% identity. A unique feature of both invertebrate and vertebrate VSPs is that depolarization induces phosphoinositide phosphatase activity through coupling of VSD to the phosphatase region (7-9). The enzymatic activity of Ci-VSP increases in the range of membrane potentials, from Ϫ80 to 100 mV, correlating with the extent of voltage sensor movement (7-8). Ci-VSP most likely operates as a monomer as shown by a study of single molecule imaging in heterologous expression system (10). The linker region between the VSD and ...

A human phospholipid phosphatase activated by a transmembrane control module

“…Given the striking structural similarity of the intracellular CD of VSPs to PTEN ( 15,27 ), the distinct enzymatic activities may appear surprising. However, the catalytic CX 5 R motif of hVSP (CKGGKGR) differs in a single position from PTEN (CK A GKGR).…”

“…However, the catalytic CX 5 R motif of hVSP (CKGGKGR) differs in a single position from PTEN (CK A GKGR). 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 ).…”

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

“…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). When this residue in Ci-VSP was mutated to the corresponding amino acid of the PTEN active site, the mutated Ci-VSP still showed both 5-and 3-phosphatase activities toward polyphosphoinositides (14).…”

“…This enzyme domain is homologous to tumor suppressor phosphatase and tensin homolog (PTEN), a phosphoinositide 3-phosphatase that dephosphorylates both phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (1). Three distinctive regions of PTEN, an N-terminal phospholipidbinding motif that anchors the protein at the plasma membrane, a phosphatase domain that has the enzymatic site, and a C-terminal lipid-interacting C2 domain (9,10), are well conserved in sequence and structure in the VSPs (11)(12)(13)(14).…”

“…Propagation of the depolarization-induced conformational changes of the VSD to the cytosolic catalytic domain activates the unique voltage-activated phosphoinositide phosphatase activity (12,14,15). Unlike its analog PTEN, which has only 3-phosphatase activity (9, 16), Ci-VSP was seen initially to cleave the Significance Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] 3-phosphatase that plays important roles in cell polarization, division, and development as well as in tumor suppression.…”