Voltage sensitive phosphatases: understanding their function and expanding their potential use

Voltage sensitive phosphatases (VSPs) are proteins consisting of two distinct domains: a transmembrane voltage sensor domain (VSD) and a cytosolic phosphatase domain highly similar to the human tumor suppressor protein PTEN (phosphatase and tensin homologue deleted from chromosome 10). The first characterized VSP was isolated from the sea squirt Ciona Intestinalis, named Ci-VSP, one decade ago. Upon membrane depolarization, Ci-VSP has phosphatase activity against phosphoinositide (PI) substrates and acts predominantly as a 5-phosphatase of PI(4,5)P2 and PI(3,4,5)P3. VSPs exist in several species, including human, and although their function and physiological role are not completely understood, they have been proven excellent tools to rapidly and reversibly alter the phosphoinositide content of the plasma membrane in living cells. The first part of this work focuses on further characterizing and understanding the function of VSPs by using well established techniques. We used whole-cell patch clamping to control VSPs and total internal reflection fluorescent (TIRF) microscopy to record their activity. We first investigated the human VSP isoform 1 (hVSP1). hVSP1 lacks plasma membrane localization and has not been characterized in depth. Here we attempted to target hVSP1 to the plasma membrane and examine its enzymatic activity. We found that complete replacement of the VSD of hVSP1 with that of Ci-VSP resulted in a membrane targeted, voltage activated 5-phosphatase of PI(4,5)P2. Given the similarity of VSPs with PTEN, the chimeric protein PTENCiV had been created previously by fusing the VSD of Ci-VSP with PTEN. PTENCiV fully represents the enzymatic activity of PTEN (3-phosphatase of PI(3,4)P2 and PI(3,4,5)P3) and by being voltage regulated it comprises a powerful tool for studying this tumor suppressor. Specifically, we characterized the enzymatic activity of a novel PTEN mutation, A126G, identified from a prostate cancer patient. This mutation was found to convert the substrate specificity of PTEN from a 3- to a 5-phosphatase. VSPs are suggested to be involved in processes where pH and redox state changes are known to occur. Therefore we next investigated the effect of intracellular pH and redox state on VSPs activity. We saw that acidic pH increases the PI(4,5)P2 depletion by VSPs, while oxidation inhibits the enzymatic activity. Thus, it seems possible that pH and oxidation can, in addition to voltage, contribute to a fine modulation of VSPs. In the second part, we developed an easily applicable method to use VSPs for manipulation of PI levels without the use of patch clamping. We used different cation channels that upon activation led to membrane depolarization and consequently VSP activation. We then characterized methods to monitor PIs levels, using fluorescence microscopy or photometry. At last, we demonstrated the application of these techniques by employing PTENCiV to characterize the effect of known PTEN mutations and analyze the effect of PTEN inhibitors. In conclusion, in this work we increased our understanding regarding several aspects of VSPs activity, including hVSP1 and PTEN substrate specificity as well as the regulation of VSPs by intracellular pH and redox state. Lastly, we established an approach that allows for rapid manipulation and monitoring of PI levels in a population of cells and facilitates the study of PTEN mutations and pharmacological targeting

A human phospholipid phosphatase activated by a transmembrane control module

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)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1\u27s 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

Voltage sensitive phosphatases: understanding their function and expanding their potential use

Voltage sensitive phosphatases (VSPs) are proteins consisting of two distinct domains: a transmembrane voltage sensor domain (VSD) and a cytosolic phosphatase domain highly similar to the human tumor suppressor protein PTEN (phosphatase and tensin homologue deleted from chromosome 10). The first characterized VSP was isolated from the sea squirt Ciona Intestinalis, named Ci-VSP, one decade ago. Upon membrane depolarization, Ci-VSP has phosphatase activity against phosphoinositide (PI) substrates and acts predominantly as a 5-phosphatase of PI(4,5)P2 and PI(3,4,5)P3. VSPs exist in several species, including human, and although their function and physiological role are not completely understood, they have been proven excellent tools to rapidly and reversibly alter the phosphoinositide content of the plasma membrane in living cells. The first part of this work focuses on further characterizing and understanding the function of VSPs by using well established techniques. We used whole-cell patch clamping to control VSPs and total internal reflection fluorescent (TIRF) microscopy to record their activity. We first investigated the human VSP isoform 1 (hVSP1). hVSP1 lacks plasma membrane localization and has not been characterized in depth. Here we attempted to target hVSP1 to the plasma membrane and examine its enzymatic activity. We found that complete replacement of the VSD of hVSP1 with that of Ci-VSP resulted in a membrane targeted, voltage activated 5-phosphatase of PI(4,5)P2. Given the similarity of VSPs with PTEN, the chimeric protein PTENCiV had been created previously by fusing the VSD of Ci-VSP with PTEN. PTENCiV fully represents the enzymatic activity of PTEN (3-phosphatase of PI(3,4)P2 and PI(3,4,5)P3) and by being voltage regulated it comprises a powerful tool for studying this tumor suppressor. Specifically, we characterized the enzymatic activity of a novel PTEN mutation, A126G, identified from a prostate cancer patient. This mutation was found to convert the substrate specificity of PTEN from a 3- to a 5-phosphatase. VSPs are suggested to be involved in processes where pH and redox state changes are known to occur. Therefore we next investigated the effect of intracellular pH and redox state on VSPs activity. We saw that acidic pH increases the PI(4,5)P2 depletion by VSPs, while oxidation inhibits the enzymatic activity. Thus, it seems possible that pH and oxidation can, in addition to voltage, contribute to a fine modulation of VSPs. In the second part, we developed an easily applicable method to use VSPs for manipulation of PI levels without the use of patch clamping. We used different cation channels that upon activation led to membrane depolarization and consequently VSP activation. We then characterized methods to monitor PIs levels, using fluorescence microscopy or photometry. At last, we demonstrated the application of these techniques by employing PTENCiV to characterize the effect of known PTEN mutations and analyze the effect of PTEN inhibitors. In conclusion, in this work we increased our understanding regarding several aspects of VSPs activity, including hVSP1 and PTEN substrate specificity as well as the regulation of VSPs by intracellular pH and redox state. Lastly, we established an approach that allows for rapid manipulation and monitoring of PI levels in a population of cells and facilitates the study of PTEN mutations and pharmacological targeting

A human phospholipid phosphatase activated by a transmembrane control module

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)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1\u27s 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

Discovery and functional characterization of a neomorphic PTEN mutation

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy