PTEN Binding Research Articles

Proteomic and yeast 2-hybrid screens to identify PTEN binding partners

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor.

Hypoxia-Inducible Transgelin-2 Confers Treatment Resistance through Activation of PI3K/Akt/GSK3β Pathway in Glioblastoma

Glioblastoma (GBM) patients with wild-type IDH experience worse survival response to the standard treatment of surgery followed by radiation therapy (RT) and temozolomide (TMZ) chemotherapy compared to their mutant IDH counterparts. This treatment has remained relatively ineffective partly due to the highly invasive phenotype of GBM leading to therapeutic resistance and tumor recurrence. Hypoxia is one of the key characteristic features of GBM which results in cancer metastasis and confers treatment resistance. Therefore, it is paramount to identify targets to help overcome hypoxia-induced treatment resistance in glioblastoma. Our lab has identified transgelin-2 (TAGLN2) to be significantly upregulated in IDH-wt GBM through multiple molecular profiling studies. This study aims to understand the mechanisms by which TAGLN2 confers treatment resistance for developing additional treatments for GBM. Additionally, active drug development efforts are also underway to target TAGLN2 for circumventing these therapeutic resistance mechanisms for effective GBM therapy. RNAi-mediated TAGLN2 knockdown (KD) approach was employed to assess the functions of TAGLN2 in GBM patient-derived xenograft (PDX) cell lines. Series of in vitro functional assays were performed to assess the role of TAGLN2 in these cell lines. Cell proliferation, invasion ± RT and/or TMZ were assessed by MTS and Trans-well invasion assays. Subsequently, WB analysis of oncogenic signaling pathways was performed following Transgelin-2 KD. Co-IP assays and Biacore/SPR analyses were performed to study the binding affinity and kinetics for the interaction of PTEN with TAGLN2. Further, cells were intracranially implanted in nude mice to assess the role of TAGLN2 on tumor growth in vivo. Conditional KD of TAGLN2 reduces cell proliferation, survival and invasive potential of GBM PDX cell lines. TAGLN2 KD also improved the sensitivity of these cells to both TMZ and radiation in vitro, as assessed by proliferation, survival, clonal expansion, and invasion. Histopathological studies of human GBM tumors and mouse xenograft tumors showed elevated expression of TAGLN2 in the peri-necrotic region of the tumors indicating that TAGLN2 protein level was upregulated by hypoxia. We also show that TAGLN2 is induced in hypoxic microenvironments with GBM PDX cell lines and its overexpression may enhance cellular resistance towards conventional therapy. Subsequently, we also show that hypoxia-induced TAGLN2 activates the PI3K/Akt oncogenic pathway through binding and inhibition of PTEN. Finally, in vivo data using an orthotopic xenograft mouse model shows reduction of tumor growth with knockdown of TAGLN2. Our in vitro and in vivo xenograft studies suggest that TAGLN2 confers treatment resistance to GBM contributing to tumor recurrence. Altogether, TAGLN2 may serve as a potential therapeutically vulnerable target in GBM specifically through its role in cell survival and invasion.

Exosomal miR-23b from bone marrow mesenchymal stem cells alleviates oxidative stress and pyroptosis after intracerebral hemorrhage.

Our previous studies showed that miR-23b was downregulated in patients with intracerebral hemorrhage (ICH). This indicates that miR-23b may be closely related to the patho-physiological mechanism of ICH, but this hypothesis lacks direct evidence. In this study, we established rat models of ICH by injecting collagenase VII into the right basal ganglia and treating them with an injection of bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-23b via the tail vein. We found that edema in the rat brain was markedly reduced and rat behaviors were improved after BMSC exosomal miR-23b injection compared with those in the ICH groups. Additionally, exosomal miR-23b was transported to the microglia/macrophages, thereby reducing oxidative stress and pyroptosis after ICH. We also used hemin to mimic ICH conditions in vitro. We found that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was the downstream target gene of miR-23b, and exosomal miR-23b exhibited antioxidant effects by regulating the PTEN/Nrf2 pathway. Moreover, miR-23b reduced PTEN binding to NOD-like receptor family pyrin domain containing 3 (NLRP3) and NLRP3 inflammasome activation, thereby decreasing the NLRP3-dependent pyroptosis level. These findings suggest that BMSC-derived exosomal miR-23b exhibits antioxidant effects through inhibiting PTEN and alleviating NLRP3 inflammasome-mediated pyroptosis, thereby promoting neurologic function recovery in rats with ICH.

Flurbiprofen inhibits cell proliferation in thyroid cancer through interrupting HIP1R-induced endocytosis of PTEN

BackgroundThe incidence of thyroid cancer, a most common tumor in the endocrine system, has increased in recent years. A growing number of studies have focused on the molecular mechanisms of thyroid cancer subtypes, aiming to identify effective therapeutic targets. Endocytosis is of vital significance in the malignant development of tumors, although its involvement in thyroid cancer has been rarely reported.MethodsHIP1R expressions in thyroid cancer from the TCGA database were analyzed by UALCAN software. Thyroid epithelial and cancer cell lines were cultured in vitro. Western blotting and quantitative PCR were used to analyze protein and mRNA levels, respectively. Cell viability was measured by CCK-8 assay. Immunofluorescence staining indicated protein distribution in cell. Co-immunoprecipitation was used to study protein–protein interaction. Immunohistochemical staining was used to analyze protein expression in clinical tissues. Differences between groups were compared using the two-tailed Student’s t test, and those among three or more groups were compared by one-way or two-way ANOVA.ResultsIn the present study, HIP1R (Huntingtin Interacting Protein 1 Related) was found upregulated in thyroid cancer tissues and cell lines compared with that in the controls, while knockdown of HIP1R significantly inhibited the proliferation of thyroid cancer cells. Since HIP1R is essential for the clathrin-dependent endocytic process, we thereafter explored the effect of HIP1R on the endocytosis of thyroid cancer cells. Interestingly, knockdown of HIP1R significantly reduced the number of clathrin-coated pits (CCPs) in thyroid cancer cells. In addition, the interaction between HIP1R and PTEN (phosphatase and tensin homolog) was identified in thyroid cancer cells. Knockdown of HIP1R downregulated intracellular PTEN in thyroid cancer cells, but upregulated membrane-binding PTEN. Notably, flurbiprofen, a commonly used analgesic, significantly inhibited the proliferation of thyroid cancer cells and interfered with the interaction between HIP1R and PTEN, thereby enhancing the binding of PTEN to cell membrane. However, the proliferation inhibitory effect of flurbiprofen was attenuated when knocking down HIP1R or PTEN.ConclusionsUpregulated HIP1R in thyroid cancer cells promotes cell proliferation and mediates the endocytosis of PTEN. Flurbiprofen may exert an anti-tumor effect on thyroid cancer by blocking the interaction between HIP1R and PTEN.

TAT delivery of a PTEN peptide inhibitor has direct cardioprotective effects and improves outcomes in rodent models of cardiac arrest.

We have recently shown that pharmacologic inhibition of PTEN significantly increases cardiac arrest survival in a mouse model, however, this protection required pretreatment 30 min before the arrest. To improve the onset of PTEN inhibition during cardiac arrest treatment, we have designed a TAT fused cell-permeable peptide (TAT-PTEN9c) based on the C-terminal PDZ binding motif of PTEN for rapid tissue delivery and protection. Western blot analysis demonstrated that TAT-PTEN9c peptide significantly enhanced Akt activation in mouse cardiomyocytes in a concentration- and time-dependent manner. Mice were subjected to 8 min asystolic arrest followed by CPR, and 30 mice with successful CPR were then randomly assigned to receive either saline or TAT-PTEN9c treatment. Survival was significantly increased in TAT-PTEN9c-treated mice compared with that of saline control at 4 h after CPR. The treated mice had increased Akt phosphorylation at 30 min resuscitation with significantly decreased sorbitol content in heart or brain tissues and reduced release of taurine and glutamate in blood, suggesting improved glucose metabolism. In an isolated rat heart Langendorff model, direct effects of TAT-PTEN9c on cardiac function were measured for 20 min following 20 min global ischemia. Rate pressure product was reduced by >20% for both TAT vehicle and nontreatment groups following arrest. Cardiac contractile function was completely recovered with TAT-PTEN9c treatment given at the start of reperfusion. We conclude that TAT-PTEN9c enhances Akt activation and decreases glucose shunting to the polyol pathway in critical organs, thereby preventing osmotic injury and early cardiovascular collapse and death.NEW & NOTEWORTHY We have designed a cell-permeable peptide, TAT-PTEN9c, to improve cardiac arrest survival. It blocked endogenous PTEN binding to its adaptor and enhanced Akt signaling in mouse cardiomyocytes. It improved mouse survival after cardiac arrest, which is related to improved glucose metabolism and reduced glucose shunting to sorbitol in critical organs.

MicroRNA-25-3p promotes cisplatin resistance in Non-small-cell lung carcinoma (NSCLC) through adjusting PTEN/PI3K/AKT route

ABSTRACT MicroRNAs exert crucial effects in the drug resistance. The purpose of this research was to investigate the miR-25-3p effects on DDP resistance in NSCLC. We used RT-qPCR to evaluate the expression of miR-25-3p. Cell growth was determined using MTS assay. Cellular bio-activity was analyzed via Colony formation, Annexin V/PI, and Transwell assay. Luciferase reporter assay was used to determine miR-25-3p and PTEN binding. Western blot was used to determine PTEN, PI3K, p-AKT/AKT expression. In-vivo study was used to determine the effects of miR-25-3p on the tumor growth. Expression of miR-25-3p is increased in NSCLC cisplatin resistant A549 and H1299 cells. Furthermore, miR-25-3p mimic enhanced drug resistance, and accelerated cell invasion and metastasis. Moreover, miR-25-3p mimic resulted in the activation of PTEN/PI3K/AKT pathway. However, miR-25-3p inhibitors exhibited the opposite trend. We further identified PTEN as a potential target of miR-25-3p. PTEN knockout promoted cisplatin resistance, while PTEN mimic displayed opposite effects. Interestingly, miR-25-3p further boosted cisplatin resistance cells in vivo, and miR-25-3p inhibitors reduced the in-vivo tumor volume. MiR-25-3p/PTEN/PI3K/AKT axis might accelerate DDP tolerance in NSCLC, which may serve as a potential target for chemotherapy resistance in NSCLC.

Abstract 216: Tat Delivery of a Pten Peptide Inhibitor Has Direct Cardioprotective Effects and Improves Outcomes in Rodent Models of Cardiac Arrest

Introduction: We have recently shown that pharmacologic inhibition of PTEN significantly increases cardiac arrest survival in a mouse model, however, this protection required pretreatment 30 min prior to the arrest. To improve the onset of PTEN inhibition during cardiac arrest treatment, we have designed a TAT fused cell-permeable peptide (TAT-PTEN9c) for rapid tissue delivery and protection. Hypothesis: We hypothesized that TAT-PTEN9c interferes with the endogenous PTEN binding to its regulatory proteins, resulting in reduced PTEN activity, improved mouse survival and cardiac functional recovery. The improved survival is in part due to enhanced glycolysis and reduced shunting to polyol pathway and osmotic injury in heart and brain. Methods: TAT-PTEN9c (7.5 mg/kg) was given intravenously after CA in mouse to determine protective effects of the treatment on survival and heart function. Western blot was used to determine the efficacy of TAT-PTEN9c for enhancing Akt and PDH E1α activity. The effect of TAT-PTEN9c on sorbitol accumulation in tissues was measured by spectrophotometer using NAD as substrate. Direct effect of TAT-PTEN9c treatment on cardiac function were also measured in Langendorff model of isolated rat heart. Results: In the mouse model of cardiac arrest, survival was significantly increased in the TAT-PTEN9c treated group compared to saline controls at 4 h after CPR. The treated mice had increased Akt phosphorylation and pyruvate dehydrogenase dephosphorylation at R30 min in heart tissues with significantly decreased sorbitol content and reduced release of taurine and glutamate into blood, suggesting improved metabolic recovery and glucose utilization. For the isolated heart model, RPP was reduced by 25% for non-treatment groups following arrest. With TAT-PTEN9c treatment, cardiac contractile function was completely recovered. TAT-PTEN9c significantly increased lactate production at 20 min of reperfusion, indicating increased glycolysis. Conclusion: TAT-PTEN9c enhances Akt and pyruvate dehydrogenase activity and decrease glucose shunting to the polyol pathway in critical organs, preventing osmotic injury and early cardiovascular collapse and death.

DJ-1-binding compound B enhances Nrf2 activity through the PI3-kinase-Akt pathway by DJ-1-dependent inactivation of PTEN

DJ-1 was identified as an oncogene and also as a causative gene for a familial form of Parkinson disease (PD). DJ-1 plays various roles in anti-oxidative stress response. Superfluous oxidation of DJ-1 at cysteine residue 106 (C106), an inactive form of DJ-1, was observed in PD patients. DJ-1-binding compound B, which specifically bound to the C106 region of DJ-1, has been isolated and it has been shown to prevent oxidative stress-induced cell death through maintaining active forms of DJ-1 by inhibiting its superfluous oxidation. The molecular mechanism of the action of compound B, however, has not been fully elucidated. In this study, we found that compound B stimulated transcriptional activity of Nrf2 in H2O2-treated SH-SY5Y cells by inhibiting its degradation through the ubiquitin-proteasome system. Although Keap 1 is a major negative regulator of Nrf2, compound B strongly increased Nrf2 activity in Keap1-mutant A549 cells but not in PTEN-null PC3 and PTEN-knockout SH-SY5Y cells. Furthermore, treatment of cells with inhibitors of the PI3-kinase/Akt pathway inhibited the effect of compound B, and compound B increased the binding of PTEN to DJ-1 and decreased lipid phosphatase activity of PTEN concomitantly with increased oxidation of PTEN, an inactive form of PTEN. These results suggest that compound B enhances transcriptional activity of Nrf2 under an oxidative stress condition in a Keap1-independent manner and that its activity is elicited by activation of the PI3Kinase/Akt pathway with DJ-1-dependent inactivation of PTEN, leading to protection of oxidative stress-induced cell death.

Abstract 276: TAT-PTEN9c Targets Heart Directly to Improve Survival From Cardiac Arrest

Introduction: It is well established that hypothermia enhances cardiac arrest (CA) survival due, in part, to a mechanism of enhanced Akt activation. In this study we investigate whether the biosynthetic PTEN inhibitor (TAT-PTEN9c) provides therapeutic protection on survival due to direct effects on heart. Hypothesis: We hypothesized that TAT-PTEN9c interferes with endogenous PTEN binding at its cell membrane adaptor and leads to enhanced Akt activation, mouse survival and cardiac functional recovery. The improved CA survival is due in part to the improved contractile functional recovery of heart by TAT-PTEN9c. We also tested if the treatment targets the heart directly to enhanced recovery in isolated perfused rat heart after a period of global arrest. Methods: TAT-PTEN9c (7.5 mg/kg) was given intravenously (IV) after CA in mouse to determine protective effects of the treatment on survival and heart function. Western blot was used to determine the efficacy of TAT-PTEN9c for Akt activation. In the isolated rat heart, direct effect of treatment (1-10 μm) on cardiac function (HR, LVDP, RPP) were measured for 20 min following 20 min global ischemia. Results: In the mouse model of cardiac arrest, survival was significantly increased in the TAT-PTEN9c treated group compared to saline controls at 4 h (10/15, 67% vs. 6/15, 40%, P < 0.05) after CPR. TAT-PTEN9c improved MAP at both R30 min and R2h. The treated mice had increased Akt phosphorylation at R15 min in both heart and brain tissues with significantly decreased sorbitol content and reduced release of taurine and glutamate into blood, suggesting improved metabolic recovery and glucose utilization. For the isolated heart model, RPP was reduced by 25% for both TAT vehicle and non-treatment groups following arrest. With TAT-PTEN9c treatment, cardiac contractile function was completely recovered (pre-arrest RPP 29.1k+/- 4.8; post-arrest 28.0K +/- 5.0). Conclusion: TAT-PTEN9c enhances Akt activation and decrease glucose shunting to the polyol pathway in critical organs, preventing osmotic injury and early cardiovascular collapse and death. The improved survival by this treatment is in part due to its direct effect on heart functional recovery.

Precise Immunodetection of PTEN Protein in Human Neoplasia.

PTEN is a major tumor-suppressor protein whose expression and biological activity are frequently diminished in sporadic or inherited cancers. PTEN gene deletion or loss-of-function mutations favor tumor cell growth and are commonly found in clinical practice. In addition, diminished PTEN protein expression is also frequently observed in tumor samples from cancer patients in the absence of PTEN gene alterations. This makes PTEN protein levels a potential biomarker parameter in clinical oncology, which can guide therapeutic decisions. The specific detection of PTEN protein can be achieved by using highly defined anti-PTEN monoclonal antibodies (mAbs), characterized with precision in terms of sensitivity for the detection technique, specificity for PTEN binding, and constraints of epitope recognition. This is especially relevant taking into consideration that PTEN is highly targeted by mutations and posttranslational modifications, and different PTEN protein isoforms exist. The precise characterization of anti-PTEN mAb reactivity is an important step in the validation of these reagents as diagnostic and prognostic tools in clinical oncology, including their routine use in analytical immunohistochemistry (IHC). Here, we review the current status on the use of well-defined anti-PTEN mAbs for PTEN immunodetection in the clinical context and discuss their potential usefulness and limitations for a more precise cancer diagnosis and patient benefit.

Abstract 3461: Characterizing the PTEN - p85alpha interaction

Abstract Introduction: The phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in regulating cell growth and cell survival and is frequently deregulated in cancer cells. p85α regulates the p110α lipid kinase, and also stabilizes and stimulates PTEN, the lipid phosphatase that downregulates this pathway. We set out to identify residues in both PTEN and p85α that mediate their interaction to better understand the regions important in mediating binding. Experimental Procedures: We previously showed that the BH domain of p85α is sufficient to mediate binding to PTEN. In this work, a deletion analysis and point mutations were used to mutate each of PTEN and the p85α BH domain to identify residues important for binding, determined using a pull-down analysis. Mutations in the p85α BH domain and in PTEN that reduced their interaction were then used as input data and docking software was used to model possible interaction interfaces for the two the proteins. Further mutagenesis and follow-up binding experiments provided support for our model of the PTEN - p85α BH domain complex. Results: We identified key residues responsible for mediating PTEN - p85α complex formation. Based on these experimental results, a docking model for the PTEN - p85α BH domain complex was developed that is consistent with the known binding interactions for both PTEN and p85α. This model involves extensive side-chain and peptide backbone contacts between both the PASE (R84, Q87, Y88, E91, E99) and C2 (R189, P190, Q219, C250, D252) domains of PTEN and the p85α BH domains with a buried surface area of 1211 Å2 (PTEN - bovine p85α BH) and 1366 Å2 (PTEN - human p85α BH). The p85α BH domain residues that directly contact PTEN in the two docking models were not identical, however both models implicated p85α residues E212, Q221, K225, R228, H234 and W237. The majority of these p85α BH domain residues were confirmed experimentally as important for PTEN binding. We also verified experimentally the importance of PTEN-E91 in mediating interaction with the p85α BH domain. Conclusions: These results shed new light on the mechanism of PTEN binding and regulation by p85α. Citation Format: Jeremy Marshall, Paul Mellor, Xuan Ruan, Dielle Whitecross, Stanley Moore, Deborah Anderson. Characterizing the PTEN - p85alpha interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3461.

Insight into the PTEN - p85α interaction and lipid binding properties of the p85α BH domain.

The phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in regulating cell growth and cell survival and is frequently deregulated in cancer cells. p85α regulates the p110α lipid kinase, and also stabilizes and stimulates PTEN, the lipid phosphatase that downregulates this pathway. In this report, we determined that the p85α BH domain binds several phosphorylated phosphoinositide lipids, an interaction that could help localize p85α to membranes rich in these lipids. We also identified key residues responsible for mediating PTEN – p85α complex formation. Based on these experimental results, a docking model for the PTEN – p85α BH domain complex was developed that is consistent with the known binding interactions for both PTEN and p85α. This model involves extensive side-chain and peptide backbone contacts between both the PASE and C2 domains of PTEN with the p85α BH domains. The p85α BH domain residues shown to be important for PTEN binding were p85α residues E212, Q221, K225, R228 and H234. We also verified experimentally the importance of PTEN-E91 in mediating the interaction with the p85α BH domain. These results shed new light on the mechanism of PTEN regulation by p85α.

Abstract 323: A Peptide Inhibitor of PTEN Improves Mouse Survival After Cardiac Arrest

Introduction: Prior works from our laboratory found that cooling protection after cardiac arrest is mediated by enhanced Akt activation and in cardiomyocyte the cooling protection can be reproduced using PTEN chemical inhibitor. The current study extend these works by designing a cell-permeable peptide, TAT-PTEN9c, which is more specific for PTEN. Hypothesis: We hypothesized that TAT-PTEN9c interferes with endogenous PTEN binding to cell membrane adaptor resulting in increased Akt activation, enhanced glucose utilization and improved mouse survival after cardiac arrest. Methods: Mouse cardiomyocytes were isolated from 1-3 day old mouse pups. Western blot was used to determine the efficacy of TAT-PTEN9c for Akt activation. The effect of TAT-PTEN9c on mouse survival after cardiac arrest was determined in a mouse model. TAT-PTEN9c (7.5 mg/kg) was given intravenously (IV) after CPR. As a measure of impaired glucose utilization, sorbitol content in heart and brain was determined by a fluorescence assay of NADH formation using sorbitol dehydrogenase and NAD + . Results: TAT-PTEN9c peptide enhanced Akt activation in mouse cardiomyocytes in a concentration-dependent manner. Akt phosphorylation was observed at 1 μM and further increased with 10 μM TAT-PTEN9c. TAT-PTEN9c blocked the binding of endogenous PTEN to MAGI2 in a co-immunoprecipitation assay, while TAT-PTEN3a control had no inhibitory effect. In a mouse model of cardiac arrest, survival was significantly increased in the TAT-PTEN9c treated group compared to saline controls at 4 h (10/15, 67% vs. 6/15, 40%, P < 0.05) after CPR. TAT-PTEN9c improved MAP at both R30 min and R2h. The treated mice had increased Akt phosphorylation at R15 min in both heart and brain tissues with significantly decreased sorbitol content and reduced release of taurine and glutamate into blood, suggesting improved metabolic recovery and glucose utilization. Conclusion: TAT-PTEN9c can be used after CPR in a mouse SCA model to rapidly enhance Akt activation and decrease glucose shunting to the polyol pathway in critical organs, preventing osmotic injury and early cardiovascular collapse and death.

Mutual inhibition between PTEN and PIP3 generates bistability for polarity in motile cells

Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. However, the mechanism that causes this spatial separation between the two molecules is unknown. Here we develop a method to manipulate PIP3 levels in living cells and used it to show PIP3 suppresses the membrane localization of PTEN. Single-molecule measurements of membrane-association and -dissociation kinetics and of lateral diffusion reveal that PIP3 suppresses the PTEN binding site required for stable PTEN membrane binding. Mutual inhibition between PIP3 and PTEN provides a mechanistic basis for bistability that creates a PIP3-enriched/PTEN-excluded state and a PTEN-enriched/PIP3-excluded state underlying the strict spatial separation between PIP3 and PTEN. The PTEN binding site also mediates the suppression of PTEN membrane localization in chemotactic signaling. These results illustrate that the PIP3-PTEN bistable system underlies a cell’s decision-making for directional movement irrespective of the environment.

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.

The Dietary Supplement Chondroitin-4-Sulfate Exhibits Oncogene-Specific Pro-tumor Effects on BRAF V600E Melanoma Cells

Dietary supplements such as vitamins and minerals are widely used in the hope of improving health but may have unidentified risks and side effects. In particular, a pathogenic link between dietary supplements and specific oncogenes remains unknown. Here we report that chondroitin-4-sulfate (CHSA), anatural glycosaminoglycan approved as a dietarysupplement used for osteoarthritis, selectively promotes the tumor growth potential of BRAF V600E-expressing human melanoma cells in patient- and cell line-derived xenograft mice and confers resistance to BRAF inhibitors. Mechanistically, chondroitin sulfate glucuronyltransferase (CSGlcA-T) signals through its product CHSA to enhance casein kinase 2 (CK2)-PTEN binding and consequent phosphorylation and inhibition of PTEN, which requires CHSA chains and is essential to sustain AKT activation in BRAF V600E-expressing melanoma cells. However, this CHSA-dependent PTEN inhibition is dispensable in cancer cells expressing mutant NRAS or PI3KCA, which directly activate the PI3K-AKT pathway. These results suggest that dietary supplements may exhibit oncogene-dependent pro-tumor effects.

Structural and Energetic Details of the Binding of PTEN to Phosphatidylinositol Phosphate-Containing Membranes through Molecular Simulations

Structural and Energetic Details of the Binding of PTEN to Phosphatidylinositol Phosphate-Containing Membranes through Molecular Simulations

PTEN Regulates Glucose Transporter Recycling by Impairing SNX27 Retromer Assembly

SummaryThe tumor suppressor PTEN executes cellular functions predominantly through its phosphatase activity. Here we identified a phosphatase-independent role for PTEN during vesicular trafficking of the glucose transporter GLUT1. PTEN physically interacts with SNX27, a component of the retromer complex that recycles transmembrane receptors such as GLUT1 from endosomes to the plasma membrane. PTEN binding with SNX27 prevents GLUT1 accumulation at the plasma membrane because of defective recycling and thus reduces cellular glucose uptake. Mechanistically, PTEN blocks the association of SNX27 with VPS26 and thereby hinders assembly of a functional retromer complex during the receptor recycling process. Importantly, we found a PTEN somatic mutation (T401I) that is defective in disrupting the association between SNX27 and VPS26, suggesting a critical role for PTEN in controlling optimal GLUT1 levels at the membrane to prevent tumor progression. Together, our results reveal a fundamental role of PTEN in the regulation of the SNX27 retromer pathway, which governs glucose transport and might contribute to PTEN tumor suppressor function.

PTEN Binding Mechanism with Compositionally Diverse Lipid Model Membranes

PTEN is one of the most frequent mutated tumor suppressor proteins in human cancer. This project investigates how PTEN interacts with lipid model membranes of different composition and morphology. We hypothesize that PTEN binds synergistically to the plasma membrane (PM) by interacting with phosphatidylinositol-(4,5)-bisphosphate(PI(4,5)P2) through its PBM domain as well as non-specifically, electrostatically through its C2 domain with anionic lipids. While the currently accepted model is that PTEN's C2 domain interacts with phosphatidylserine (PS), we hypothesize that the C2 domain can interact with any anionic lipid, as long as this lipid has a sufficiently high local concentration. PI(4,5)P2 is present in the PM at about 1%, while PS is found at about 30%. We observed in model membrane studies that PI(4,5)P2 and PS have only a limited tendency to co-localize in a domain. In contrast, phosphatidylinositol (PI) (about 8% of PM lipids), aids in the formation of PI(4,5)P2 enriched domains through headgroup hydrogen bond formation. A PI/PI(4,5)P2 domain would be an excellent platform for PTEN membrane interaction, suggesting that PI is a better second binding partner than PS. We are using stopped-flow fluorescence spectrophotometry as an ensemble technique that provides us with information about the association and dissociation kinetics of PTEN when interacting with lipid model membranes of different composition. Using this technique we have found that PTEN binds to pure PS and PI vesicles with a similar binding constant. We see synergistic binding to 30%PS/5%PI(4,5)P2 as well as 30%PI 5%PI(4,5)P2. Additionally, we are utilizing single molecule TIRF microscopy to track PTEN on model membranes of different composition. From these data, we determine the PTEN diffusion coefficient, hopping frequency and dwell times. In the aggregate, this allows us to describe in detail PTEN binding mechanisms with differently composed lipid model membranes.

Mechanistic insights into the role of truncating PREX2 mutations in melanoma

ABSTRACTPREX2 is a PTEN binding protein that is significantly mutated in melanoma and pancreatic ductal adenocarcinoma. We recently reported the molecular mechanism of tumorigenesis associated with PREX2 mutations: truncating PREX2 mutations activate its RAC1 guanine nucleotide exchanger activity leading to increased PI3K/AKT signaling and enhanced cell proliferation.