This article is available online at http://www.jlr.org domain (VSD) homologous to the voltage sensors of voltage-gated ion channels and an intracellular C-terminal catalytic domain (CD) with high similarity to the tumor suppressor lipid phosphatase, PTEN ( Fig.1A ) ( 1 ). The enzymatic activity of the CD is controlled by the VSD via an intramolecular conformational switch ( 4, 5 ). At typical negative resting voltages, the CD is inactive and is rapidly activated at more-positive (depolarized) voltages. The VSP homologs characterized to date are phosphoinositide 5-phosphatases that degrade the major signaling phospholi pids PI(4,5)P 2 and PI(3,4,5)P 3 ( 2 ). Both phosphoinositides have key signaling roles in many cellular processes, including cell proliferation and differentiation, cytoskeletal dynamics, membrane traffi cking, and control of ion channels ( 6, 7 ). Although little is known to date about the biological functions of VSPs, the ubiquitous roles of phosphoinositides and of electrical signaling suggest a potential impact on a large spectrum of cellular processes.The principle of operation of VSPs was initially demonstrated for Ci-VSP, the prototypic VSP from the invertebrate chordate Ciona intestinalis . Subsequently, functional vertebrate VSPs have been identifi ed in fi shes and amphibia ( 8, 9 ). The VSP gene is also conserved in mammals ( 10 ). In general, there appears to be one VSP homolog in mammalian genomes; in the human genome, however, there are two expressed homologs, TPTE and TPTE2 (also termed TPIP) and additional pseudo-genes ( 10, 11 ). TPTE conforms to the architecture of VSPs, but it lacks phosphatase activity due to amino acid exchanges in the catalytic CX 5 R motif in the P-loop of the phosphatase domain The recent discovery of voltage-sensitive phosphatases (VSPs) ( 1 ) established a novel molecular principle of electrochemical coupling: VSPs directly mediate the degradation of phosphoinositides in response to depolarization of the membrane potential ( 2, 3 ). These so-far-unique electro-enzymes consist of a transmembrane voltage sensor
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 welldefined 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.functional genomics | PTEN | tumor suppressor
Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P2 and PI(3,4,5)P3 levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P3. Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P2 and PI(3,4,5)P3 levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells.
PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN’s substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2867-z) contains supplementary material, which is available to authorized users.
Voltage-sensitivephosphatases (VSPs) are proteins that dephosphorylate phosphoinositides (PIs) upon membrane voltage dependent activation. Particularly, Ci-VSP (Ciona intestinalis Voltage-Sensitive Phosphatase) is a 5-phosphatase of PI(4,5)P 2 and PI(3,4,5)P 3 and has so far been studied by electrophysiological means.Here we developed more broadly applicable methods of membrane depolarization and therefore phosphatase activation. First, depolarization was achieved by coexpression of the K þ channels TASK and KCNQ and elevation of extracellular potassium concentration. Second, we utilized the capsaicinactivated TRPV1 channel, and last the photosensitive Channel Rhodopsin, ChR2, in order to activate Ci-VSP. Cultured cell lines were transfected with the different channels, Ci-VSP and sensors for PIs, e.g. the tubby and the PLCd1-PH domain, each tagged with a fluorescent protein. Confocal microscopy was used to visualize and quantify the translocation of the PI-sensors from the membrane to the cytoplasm after the dephosphorylation of PIs. All three methods resulted in successful activation of Ci-VSP, with TRPV1 and ChR2 mediated depolarization to give the most robust results. Specifically, the ChR2 activation provides a very rapid response without the need of solution exchanges, simplifying the experimental procedures. To conclude, we suggest a series of methods that allow the manipulation of PI levels as well as the study of VSPs in living cells without electrophysiological instrumentation. This work was supported by a grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to CRH and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to DO. 2256-Pos Board B275 Ion Induced Changes in Phosphoinositide Monolayers at Phisiological Concentrations
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