PTEN Lipid Research Articles

Cancer and Autism: How PTEN Mutations Degrade Function at the Membrane and Isoform Expression in the Human Brain

Mutations causing loss of PTEN lipid phosphatase activity can promote cancer, benign tumors (PHTS), and neurodevelopmental disorders (NDDs). Exactly how they preferentially trigger distinct phenotypic outcomes has been puzzling. Here, we demonstrate that PTEN mutations differentially allosterically bias P loop dynamics and its connection to the catalytic site, affecting catalytic activity. NDD-related mutations are likely to sample conformations of the functional wild-type state, while sampled conformations for the strong, cancer-related driver mutation hotspots favor catalysis-primed conformations, suggesting that NDD mutations are likely to be weaker, and our large-scale simulations show why. Prenatal PTEN isoform expression data suggest exons 5 and 7, which harbor NDD mutations, as cancer-risk carriers. Since cancer requires more than a single mutation, our conformational and genomic analysis helps discover how same protein mutations can foster different clinical manifestations, articulates a role for co-occurring background latent driver mutations, and uncovers relationships of splicing isoform expression to life expectancy.

PTEN is both an activator and a substrate of chaperone-mediated autophagy.

PTEN is a crucial negative regulator of the INS/PI3K/AKT pathway and is one of the most commonly mutated tumor suppressors in cancer. Global overexpression (OE) of PTEN in mice shifts metabolism to favor oxidative phosphorylation over glycolysis, reduces fat mass, and extends the lifespan of both sexes. We demonstrate that PTEN regulates chaperone-mediated autophagy (CMA). Using cultured cells and mouse models, we show that PTEN OE enhances CMA, dependent upon PTEN's lipid phosphatase activity and AKT inactivation. Reciprocally, PTEN knockdown reduces CMA, which can be rescued by inhibiting class I PI3K or AKT. Both PTEN and CMA are negative regulators of glycolysis and lipid droplet formation. We show that suppression of glycolysis and lipid droplet formation downstream of PTEN OE depends on CMA activity. Finally, we show that PTEN protein levels are sensitive to CMA and that PTEN accumulates in lysosomes with elevated CMA. Collectively, these data suggest that CMA is both an effector and a regulator of PTEN.

Chemical and structural approaches to investigate PTEN function and regulation.

Phosphatase and tensin homolog is a lipid phosphatase that serves as the major negative regulator of the PI3K/AKT pathway. It catalyzes the 3'-specific dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to generate PIP2. PTEN's lipid phosphatase function depends on several domains, including an N-terminal segment spanning the first 24 amino acids, which results in a catalytically impaired enzyme when mutated. Furthermore, PTEN is regulated by a cluster of phosphorylation sites located on its C-terminal tail at Ser380, Thr382, Thr383, and Ser385, which drives its conformation from an open to a closed autoinhibited but stable state. Herein, we discuss the protein chemical strategies we used to reveal the structure and mechanism of how PTEN's terminal regions govern its function.

Phosphatase and Tensin Homology Deleted on Chromosome 10 Inhibitors Promote Neural Stem Cell Proliferation and Differentiation.

Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor gene. Its encoded protein has phosphatase and lipid phosphatase activities, which regulate the growth, differentiation, migration, and apoptosis of cells. The catalytic activity of PTEN is crucial for controlling cell growth under physiological and pathological conditions. It not only affects the survival and proliferation of tumor cells, but also inhibits a variety of cell regeneration processes. The use of PTEN inhibitors is being explored as a potentially beneficial therapeutic intervention for the repair of injuries to the central nervous system. PTEN influences the proliferation and differentiation of NSCs by regulating the expression and phosphorylation of downstream molecular protein kinase B (Akt) and the mammalian target of rapamycin (mTOR). However, the role of PTEN inhibitors in the Akt/mTOR signaling pathway in NSC proliferation and differentiation is unclear. Dipotassium bisperoxo (picolinoto) oxovanadate (V) [bpv(pic)] is a biologically active vanadium compound that blocks PTEN dephosphorylation and suppresses its activity, and has been used as a PTEN lipid phosphatase inhibitor. Here, bpv(pic) intervention was found to significantly increase the number of rat NSCs, as determined by bromodeoxyuridine staining and the cell counting kit-8, and to increase the percentage of neurons undergoing differentiation, as shown by immunofluorescence staining. Bpv(pic) intervention also significantly increased PTEN and mTOR expression, as shown by real-time PCR analysis and western blotting. In conclusion, PTEN inhibitor bpv(pic) promotes the proliferation and differentiation of NSCs into neurons.

The role of PI3'-lipid signalling in melanoma initiation, progression and maintenance.

Phosphatidylinositol-3'-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3' hydroxyl (OH) of the inositol ring of phosphatidylinositides (PI). Through their downstream effectors, PI3K generated lipids (PI3K-lipids hereafter) such as PI(3,4,5)P3 and PI(3,4)P2 regulate myriad biochemical and biological processes in both normal and cancer cells including responses to growth hormones and cytokines; the cell division cycle; cell death; cellular growth; angiogenesis; membrane dynamics; and autophagy and many aspects of cellular metabolism. Engagement of receptor tyrosine kinase by their cognate ligands leads to activation of members of the Class I family of PI3'-kinases (PI3Kα, β, δ & γ) leading to accumulation of PI3K-lipids. Importantly, PI3K-lipid accumulation is antagonized by the hydrolytic action of a number of PI3K-lipid phosphatases, most notably the melanoma suppressor PTEN (lipid phosphatase and tensin homologue). Downstream of PI3K-lipid production, the protein kinases AKT1-3 are believed to be key effectors of PI3'-kinase signalling in cells. Indeed, in preclinical models, activation of the PI3K→AKT signalling axis cooperates with alterations such as expression of the BRAFV600E oncoprotein kinase to promote melanoma progression and metastasis. In this review, we describe the different classes of PI3K-lipid effectors, and how they may promote melanomagenesis, influence the tumour microenvironment, melanoma maintenance and progression to metastatic disease. We also provide an update on both FDA-approved or experimental inhibitors of the PI3K→AKT pathway that are currently being evaluated for the treatment of melanoma either in preclinical models or in clinical trials.

Abstract 2534: Underlying mechanism of response to neddylation inhibition in a subset of glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults with a 5-year survival rate of ~3%. The ubiquitin/proteasome system maintains intracellular homeostasis via degradation of unwanted proteins. Bortezomib is a first-in-class, non-specific proteasome inhibitor exploiting this system, although it has poor penetration across the blood brain barrier, and its lack of specificity is accompanied by adverse events. Neddylation is a specific pathway within the ubiquitin/proteasome system that is overactive in glioblastoma (GBM), and whose upregulation has been associated with glioma progression and worse survival. Pevonedistat is a first-in-class small-molecule neddylation inhibitor shown to impact protein degradation, leading to elevated abundance of some tumor suppressor proteins (Wee1, others), which then inhibit growth of GBM cells in culture and orthotopic xenografts. Because the molecular heterogeneity within and across GBM patients obscures therapeutic targets and obfuscates signals of efficacy in clinical trials, we propose the use of molecular “signatures of vulnerability” to targeted agents in subsets of preclinical models. We and others have shown that pevonedistat interferes with the growth of multiple types of cancers, including GBM, but the determinants of vulnerability are not fully understood. Here, we report a selective vulnerability to pevonedistat in a subset of GBM, specifically, instances with mutations or copy number deletions of PTEN are associated with de novo resistance to pevonedistat. Time-course studies of sensitive and non-sensitive GBM cells using transcriptomics and proteomics/phosphoproteomics enable independent discovery and testing for determinants of response to pevonedistat. Our results demonstrate that in GBM, resistance to pevonedistat is driven by reduced PTEN-chromatin binding (loss-of-function or lower expression) that is also independent of PTEN's lipid phosphatase activity (i.e., PI3K/AKT signaling). Across 25 glioma cell lines, we found that PTEN signaling, DNA replication, and chromatin instability pathways are the most significant differentiators between pevonedistat sensitive vs. non-sensitive models. In GBM models with modest to low sensitivity to pevonedistat, TOP2A expression was elevated. Combination treatment with the TOP2A inhibitor, etoposide, proved synergistic with pevonedistat. We report, for the first time, that PTEN status both serves as a novel biomarker for GBM sensitivity to pevonedistat and reveals a synergistic vulnerability of TOP2A inhibitors in combination with pevonedistat. Paired use of GBM PDX models of varying sensitivity with drug development testing allows the advancement of a promising agent as well as a patient-enrollment “signature of vulnerability” likely to increase the likelihood of demonstrating therapeutic efficacy in early clinical trials. Citation Format: Shayesteh Ferdosi, Brett Taylor, Matthew Lee, Nanyun Tang, Sen Peng, Rita Bybee, George Reid, Lauren Hartman, Krystine Garcia-Mansfield, Ritin Sharma, Patrick Pirrotte, Frank Furnari, Harshil Dhruv, Michael Berens. Underlying mechanism of response to neddylation inhibition in a subset of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2534.

PTEN and Other PtdIns(3,4,5)P3 Lipid Phosphatases in Breast Cancer.

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PtdIns(3,4,5)P3 can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P2. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

PTEN Lipid Phosphatase Activity Enhances Dengue Virus Production through Akt/FoxO1/Maf1 Signaling

Dengue virus (DENV) is an arthropod-borne viral pathogen and a global health burden. Knowledge of the DENV-host interactions that mediate virus pathogenicity remains limited. Host lipid metabolism is hijacked by DENV for virus replication in which lipid droplets (LDs) play a key role during the virus lifecycle. In this study, we reveal a novel role for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in LDs-mediated DENV infection. We demonstrate that PTEN expression is downregulated upon DENV infection through post-transcriptional regulation and, in turn, PTEN overexpression enhances DENV replication. PTEN lipid phosphatase activity was found to decrease cellular LDs area and number through Akt/FoxO1/Maf1 signaling, which, together with autophagy, enhanced DENV replication and virus production. We therefore provide mechanistic insight into the interaction between lipid metabolism and the DENV replication cycle.

Phospho Tensin Homolog in Human and Lipid Peroxides in Peripheral Blood Mononuclear Cells Following Exposure to Flavonoids

Objectives: Studies have shown that human and peripheral blood mononuclear cells (PBMCs) are mostly used for research purposes to study several biochemical endpoints. The effects of the flavonoids, genistein, kaempferol, and quercetin on phospho tensin homolog (PTEN) levels in cancer cells (i.e., breast [BT549], lung [A549]), human embryonic kidney cells (HEK293), and the levels of lipid peroxides (LP) in PBMCs were respectively investigated.Materials and methods: Cancer, kidney, and PBMCs from several donors were each exposed to each of the flavonoids at concentrations of 0, 5, 10, 15, 20, and 25 µM. Our hypotheses were that exposure of cancer and kidney cells to genistein, kaempferol, and quercetin can increase PTEN and decrease lipid peroxides in PBMCs levels respectively to better cope with oxidative stress.Results: The results indicate that the flavonoids increased total PTEN levels in a dose-dependent manner. The effect of quercetin was more pronounced followed by genistein and kaempferol. Furthermore, decreases in lipid peroxides were observed in the PBMCs for the flavonoid-treated samples compared to those exposed to flavonoids and with oxidative stress as described by Fenton’s chemistry. Levels of LP in quercetin-treated samples were lower compared to kaempferol and genistein.Conclusions: The findings suggest that the flavonoids play an important role in controlling oxidative stress in several human cells.

Oncogenic Y68 frame shift mutation of PTEN represents a mechanism of docetaxel resistance in endometrial cancer cell lines

In this study, we aimed to identify mutations of key genes associated with docetaxel resistance in nine endometrial cancer cell lines. Endometrial cancers are associated with several critical gene mutations, including PIK3A, PTEN, and KRAS. Different gene mutations in endometrial cancer cells have varied responses to anticancer drugs and cancer therapies. The most frequently altered gene in endometrioid endometrial carcinoma tumors is PTEN. PTEN protein has lipid phosphatase and protein phosphatase activity, as well as other functions in the nucleus. Although the tumor-suppressive function of PTEN has mainly been attributed to its lipid phosphatase activity, a role for PTEN protein phosphatase activity in cell cycle regulation has also been suggested. Various tumor type-specific PTEN mutations are well documented. Here, nine endometrioid endometrial cancer cell lines with PIK3A, PTEN, and KRAS gene mutations were treated with docetaxel and radiation. One mutation with a docetaxel drug-resistant effect was a truncated form of PTEN. Among PTEN mutations in endometrial cancer cells, the Y68 frame shift mutation of PTEN constitutes a major mechanism of resistance to docetaxel treatment. The molecular mechanism involves truncation of the 403 amino acid PTEN protein at amino acid 68 by the Y68 frame shift, leading to the loss of PTEN protein phosphatase and lipid phosphatase activities.

PTEN lipid phosphatase inactivation links the hippo and PI3K/Akt pathways to induce gastric tumorigenesis

BackgroundPhosphatase and tensin homolog (PTEN) is an important tumor suppressor gene, and its encoded protein has activities of both a protein phosphatase and a lipid phosphatase. However, the substitution effect of protein phosphatase activity remains unclear. PI3K/Akt is the most common pathway negatively regulated by PTEN. The Hippo and PI3K/Akt pathways have a joint effect in regulating cell proliferation and apoptosis. Therefore, how PTEN lipid phosphatase inactivation contributes to the occurrence and development of gastric cancer and the potential role of the Hippo and PI3K/Akt pathways in PTEN lipid phosphatase inactivation mediated gastric tumorigenesis remain to be explored.MethodsImmunohistochemical staining was performed to detect the expression of p-PTEN and YAP in a gastric cancer tissue microarray. Stable cell lines expressing a wild-type or dominant-negative mutant PTEN were established. The proliferation and migration of stable cells were detected by MTT, BrdU, and colony-formation, transwell assay and high content analysis in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the Hippo and Akt signaling pathways were detected by western blot. Nuclear-cytoplasm separation, immunofluorescence and coimmunoprecipitation analyses were conducted to explore the dysregulation of Hippo in the stable cell lines.ResultsPTEN lipid phosphatase inactivation strongly promoted the proliferation and migration of gastric cancer cells in vitro and tumor growth in vivo. A immunohistochemical analysis of gastric cancer tissues revealed a significant correlation between phosphorylated PTEN and nuclear YAP expression, and both were determined to be independent prognostic factors for gastric cancer. Mechanistically, PTEN lipid phosphatase inactivation abolished the MOB1-LATS1/2 interaction, decreased YAP phosphorylation and finally promoted YAP nuclear translocation, which enhanced the synergistic effect of YAP-TEAD, thus inducing cell proliferation and migration. Moreover, PTEN lipid phosphatase inactivation promoted the PI3K/Akt pathway, and disruption of YAP-TEAD-driven transcription decreased the activation of Akt in a dose-dependent manner.ConclusionsTaken together, our findings indicate that PTEN lipid phosphatase inactivation links the Hippo and PI3K/Akt pathways to promote gastric tumorigenesis and cancer development.

Patient-derived mutations within the N-terminal domains of p85\u03b1 impact PTEN or Rab5 binding and regulation

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

Protein Chemical Approaches to Understanding PTEN Lipid Phosphatase Regulation.

Since the discovery of C-tail phosphorylation of PTEN almost 20 years ago, much progress has been made in understanding its regulatory influences on the cellular function of PTEN. Phosphorylation of Ser380, Thr382, Thr383, and Ser385 drives a PTEN conformational change from an open to closed state where catalytic function is impaired, plasma membrane binding is reduced, and cellular stability is enhanced. Despite these advances, a detailed structural and mechanistic model of how these phosphorylations impact PTEN function is lacking. We discuss here several recent approaches to analyzing PTEN phosphorylation and highlight several insights that have come from this work. We also discuss remaining challenges for the PTEN regulation field and potential directions for future research.

IDO, PTEN-expressing Tregs and control of antigen-presentation in the murine tumor microenvironment.

The tumor microenvironment is profoundly immunosuppressive. This creates a major barrier for attempts to combine immunotherapy with conventional chemotherapy or radiation, because the tumor antigens released by these cytotoxic agents are not cross-presented in an immunogenic fashion. In this Focused Research Review, we focus on mouse preclinical studies exploring the role of immunosuppressive Tregs expressing the PTEN lipid phosphatase, and the links between PTEN+ Tregs and the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO has received attention because it can be expressed by a variety of human tumor types in vivo, but IDO can also be induced in host immune cells of both humans and mice in response to inflammation, infection or dying (apoptotic) cells. Mechanistically, IDO and PTEN+ Tregs are closely connected, with IDO causing activation of the PTEN pathway in Tregs. Genetic ablation or pharmacologic inhibition of PTEN in mouse Tregs destabilizes their suppressive phenotype, and this prevents transplantable and autochthonous tumors from creating their normal immunosuppressive microenvironment. Genetic ablation of either IDO or PTEN+ Tregs in mice results in a fundamental defect in the ability to maintain tolerance to antigens associated with apoptotic cells, including dying tumor cells. Consistent with this, pharmacologic inhibitors of either pathway show synergy when combined with cytotoxic agents such as chemotherapy or radiation. Thus, we propose that IDO and PTEN+ Tregs represent closely linked checkpoints that can influence the choice between immune activation versus tolerance to dying tumor cells.

Up-regulated HOTAIR induced by fatty acids inhibits PTEN expression and increases triglycerides accumulation in HepG2 cells

ABSTRACTNon-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation unrelated to excess alcohol intake, in which the hepatic expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is inhibited. Long non-coding RNA HOTAIR suppresses PTEN expression in hepatic stellate cells during liver fibrosis, and it is involved in liver lipid dysregulation. In this study, we evaluated whether the PTEN down-regulation and lipid accumulation in hepatic cells might be mediated by HOTAIR during the development of NAFLD. Free fatty acids (FFAs) treatment promoted triglyceride accumulation in HepG2 cells, significantly increasing HOTAIR expression and inhibiting PTEN expression (both at mRNA and protein levels).The effects on HOTAIR and PTEN expressions disappeared after withdrawal of the FFAs treatment. SiRNA-mediated HOTAIR knockdown prevented PTEN down-regulation and triglyceride accumulation in HepG2 cells treated with FFAs; and CAPE (an NF-κBp65 inhibitor) treatment prevented the HOTAIR up-regulation and PTEN down-regulation. FFAs could induce the up-regulation of HOTAIR in HepG2 cells probably dependent on the NF-κB signaling, and consequently suppress PTEN expression and promote triglyceride accumulation. Aberrant up-regulation of HOTAIR mediated by excessive circulating FFAs levels may be a crucial mechanism associated with liver steatosis.

Hematopoietic Stem Cell Activity Is Regulated by Pten Phosphorylation Through a Niche-Dependent Mechanism.

The phosphorylated form of Pten (p-Pten) is highly expressed in >70% of acute myeloid leukemia samples. However, the role of p-Pten in normal and abnormal hematopoiesis has not been studied. We found that Pten protein levels are comparable among long-term (LT) hematopoietic stem cells (HSCs), short-term (ST) HSCs, and multipotent progenitors (MPPs); however, the levels of p-Pten are elevated during the HSC-to-MPP transition. To study whether p-Pten is involved in regulating self-renewal and differentiation in HSCs, we compared the effects of overexpression of p-Pten and nonphosphorylated Pten (non-p-Pten) on the hematopoietic reconstitutive capacity (HRC) of HSCs. We found that overexpression of non-p-Pten enhances the LT-HRC of HSCs, whereas overexpression of p-Pten promotes myeloid differentiation and compromises the LT-HRC of HSCs. Such phosphorylation-regulated Pten functioning is mediated by repressing the cell:cell contact-induced activation of Fak/p38 signaling independent of Pten's lipid phosphatase activity because both p-Pten and non-p-Pten have comparable activity in repressing PI3K/Akt signaling. Our studies suggest that, in addition to repressing PI3K/Akt/mTor signaling, non-p-Pten maintains HSCs in bone marrow niches via a cell-contact inhibitory mechanism by inhibiting Fak/p38 signaling-mediated proliferation and differentiation. In contrast, p-Pten promotes the proliferation and differentiation of HSCs by enhancing the cell contact-dependent activation of Src/Fak/p38 signaling. Stem Cells 2016;34:2130-2144.

Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development.

The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten.

Assays to Measure PTEN Lipid Phosphatase Activity In Vitro from Purified Enzyme or Immunoprecipitates.

PTEN is a one of the most frequently mutated tumor suppressors in human cancers. It is essential for regulating diverse biological processes and through its lipid phosphatase activity regulates the PI 3-Kinase signaling pathway. Sensitive phosphatase assays are employed to study the catalytic activity of PTEN against phospholipid substrates. Here we describe protocols to assay PTEN lipid phosphatase activity using either purified enzyme (purified PTEN lipid phosphatase assay) or PTEN immunopurified from tissues or cultured cells (cellular IP PTEN lipid phosphatase assay) against vesicles containing radiolabeled PIP3 substrate.

Single Molecule Diffusion Studies of PTEN: Insights into Membrane Binding

PTEN, a tumor suppressor gene that encodes a dual specificity phosphatase that dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3), is one of the most frequent genes deleted or mutated in a wide variety of tumors. PTEN acts as an antagonist to phosphoinositide-3-kinase (PI3K) signaling, thereby affecting various cellular processes such as cell proliferation and survival. The activity of PTEN is regulated by dynamic shuttling between the cytoplasm and the plasma membrane. The membrane association of PTEN strongly depends on the composition and lateral distribution of the lipids in the membrane. Several biophysical techniques have been used to characterize PTEN-membrane interaction. Here, we use single-molecule total internal reflection fluorescence microscopy (TIRFM), which is a powerful tool to study the molecular mechanism of membrane targeted proteins. The single-molecule TIRFM allows us to observe single PTEN molecules as they dynamically associate/dissociate and laterally diffuse along the lipid bilayer membrane. PTEN lipid binding is investigated on supported lipid bilayers of binary and ternary lipid mixtures of PC with physiological relevant levels of PS, PI(4,5)P2 and PI. We tracked individual PTEN molecules and statistically determine the lateral diffusion and dwell time of PTEN on heterogeneous lipid bilayers. We find significant differences in PTEN dynamic behavior when bound to the different membrane environments. Furthermore, to gain insight into the molecular mechanisms of PTEN membrane association, we compared the lipid binding of wt PTEN, an N-terminally truncated PTEN-(Δ1–15 AA) that lacks the PI(4,5)P2 binding site, and the recently discovered, PTEN-L with a 173 AA N-terminal extension. We find profound differences in the dynamic behavior of these PTEN derivatives at the membrane. It has recently been suggested that PTEN associates as a dimer with the membrane. We find that the tendency to form membrane bound dimers varies among these PTEN variants.