Lipid Phosphatase PTEN Research Articles

Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila.

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting βHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

Dual-Specific Protein and Lipid Phosphatase PTEN and Its Biological Functions.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) encodes a 403-amino acid protein with an amino-terminal domain that shares sequence homology with the actin-binding protein tensin and the putative tyrosine-protein phosphatase auxilin. Crystal structure analysis of PTEN has revealed a C2 domain that binds to phospholipids in membranes and a phosphatase domain that displays dual-specific activity toward both tyrosine (Y), serine (S)/threonine (T), as well as lipid substrates in vitro. Characterized primarily as a lipid phosphatase, PTEN plays important roles in multiple cellular processes including cell growth/survival as well as metabolism.

A Precision B Cell-Targeted Therapeutic Approach to Autoimmunity Caused by Phosphatidylinositol 3-Kinase Pathway Dysregulation.

The inositol lipid phosphatases PTEN and SHIP-1 play a crucial role in maintaining B cell anergy and are reduced in expression in B cells from systemic lupus erythematosus and type 1 diabetes patients, consequent to aberrant regulation by miRNA-7 and 155. With an eye toward eventual use in precision medicine therapeutic approaches in autoimmunity, we explored the ability of p110δ inhibition to compensate for PI3K pathway dysregulation in mouse models of autoimmunity. Low dosages of the p110δ inhibitor idelalisib, which spare the ability to mount an immune response to exogenous immunogens, are able to block the development of autoimmunity driven by compromised PI3K pathway regulation resultant from acutely induced B cell-targeted haploinsufficiency of PTEN and SHIP-1. These conditions do not block autoimmunity driven by B cell loss of the regulatory tyrosine phosphatase SHP-1. Finally, we show that B cells in NOD mice express reduced PTEN, and low-dosage p110δ inhibitor therapy blocks disease progression in this model of type 1 diabetes. These studies may aid in the development of precision treatments that act by enforcing PI3K pathway regulation in patients carrying specific risk alleles.

Pten controls B‐cell responsiveness and germinal center reaction by regulating the expression of IgD BCR

In contrast to other B‐cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co‐express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3‐kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten‐deficient B cells expressing knock‐ins for BCR heavy and light chain genes are unable to upregulate IgD. Furthermore, in the presence of autoantigen, Pten‐deficient B cells cannot eliminate the autoreactive BCR specificity by secondary light chain gene recombination. Instead, Pten‐deficient B cells downregulate BCR expression and become unresponsive to further BCR‐mediated stimulation. Notably, we observed a delayed germinal center (GC) reaction by IgD‐deficient B cells after immunization with trinitrophenyl‐ovalbumin (TNP‐Ova), a commonly used antigen for T‐cell‐dependent antibody responses. Together, our data suggest that the activation of IgD expression by Pten/FoxO1 results in mature B cells that are selectively responsive to multivalent antigen and are capable of initiating rapid GC reactions and T‐cell‐dependent antibody responses.

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.

Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun.

The Rsu1 protein contributes to cell adhesion and migration via its association with the adaptor complex of Integrin linked kinase (ILK), PINCH, and Parvin (IPP), which binds to the cytoplasmic domain of β1 integrins joining integrins to the actin cytoskeleton. Rsu1 binding to PINCH in the IPP complex is required for EGF-induced adhesion, spreading and migration in MCF10A mammary epithelial cells. In addition, Rsu1 expression inhibits Jun kinase but is necessary for the activation of MKK4 and p38 Map kinase signaling essential for migration in MCF10A cells. The data reported here examines the links between MKK4-p38-ATF2 signaling and AKT regulation in MCF10A cells. Ectopic Rsu1 inhibited AKT1 phosphorylation while Rsu1 depletion induced AKT activation and AKT1 phosphorylation of MKK4 on serine 80, blocking MKK4 activity. Rsu1 depletion also reduced the RNA for lipid phosphatase PTEN thus implicating PTEN in modulating levels of activated AKT in these conditions. ChIP analysis of the PTEN promoter revealed that Rsu1 depletion prevented binding of ATF2 to a positive regulatory site in the PTEN promoter and the enhanced binding of cJun to a negatively regulatory PTEN promoter site. These results demonstrate a mechanism by which Rsu1 adhesion signaling alters the balance between MKK4-p38-ATF2 and cJun activation thus altering PTEN expression in MCF10A cells.

A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is predominantly a class I PI3K signaling product.

Class I phosphoinositide 3-OH kinase (PI3K) signaling is central to animal growth and metabolism, and pathological disruption of this pathway affects cancer and diabetes. However, the specific spatial/temporal dynamics and signaling roles of its minor lipid messenger, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), are not well understood. This owes principally to a lack of tools to study this scarce lipid. Here we developed a high-sensitivity genetically encoded biosensor for PI(3,4)P2, demonstrating high selectivity and specificity of the sensor for the lipid. We show that despite clear evidence for class II PI3K in PI(3,4)P2-driven function, the overwhelming majority of the lipid accumulates through degradation of class I PI3K-produced PIP3 However, we show that PI(3,4)P2 is also subject to hydrolysis by the tumor suppressor lipid phosphatase PTEN. Collectively, our results show that PI(3,4)P2 is potentially an important driver of class I PI3K-driven signaling and provides powerful new tools to begin to resolve the biological functions of this lipid downstream of class I and II PI3K.

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.

PTEN prevents the cytokine storm

Immunology An uncontrolled infection leads to sepsis, in which excessive production of proinflammatory cytokines, or a “cytokine storm,” can cause potentially fatal tissue damage and organ failure. Sisti et al. found that septic mice had increased expression of the mRNA encoding the lipid and protein phosphatase PTEN. Mice lacking PTEN in myeloid cells showed greater inflammation, tissue injury, and mortality from sepsis. PTEN activity in the nucleus of macrophages induced the production of microRNAs that targeted Myd88 mRNA, which encodes an adaptor protein required for cytokine production. Sci. Signal. 11 , eaai9085 (2018).

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.

Regulation of the Phosphatidylinositide 3-Kinase Pathway by the Lipid Phosphatase PTEN.

Featured Article: Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998;95:29–39.4 PTEN is a lipid phosphatase whose action targets phosphatidylinositol 3,4,5-trisphosphate (PIP3),5 a component of the lipid cellular membrane. Its role as a tumor suppressor was defined in 1998 in the article by Stambolic et al. highlighted here (1), which reported the extensive effects of the disruption of PTEN (phosphatase and tensin homolog) in mice. By the late 1990s, a quest for the major tumor suppressor gene within the frequently deleted region of chromosome 10 found in advanced cancers culminated in the discovery of PTEN, named based on amino acid sequence homology with the protein tyrosine phosphatases and tensin (1). The observations reported in the 1998 article contributed to the understanding of the cellular functions of PTEN and the severity of the consequences associated with PTEN loss. Homozygous …

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.

Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent.

β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb(-/-) thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4(+)CD8(+) cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.

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.

PIPPing on AKT1: How Many Phosphatases Does It Take to Turn off PI3K?

In this issue of Cancer Cell, Ooms and colleagues show that the lipid phosphatase PIPP/INPP5J, frequently inactivated in triple-negative breast cancers, functions as a tumor suppressor by specifically modulating the activity of AKT1 in the context of oncogenic PI3K signaling, leading to inhibition of metastatic dissemination.

IP3 3-kinase B prevents bone marrow failure

IP3 3-kinase B prevents bone marrow failure

Differential regulation of PTEN and Akt by TCR signal strength controls CD4+ T cell fate decisions (LYM8P.627)

Abstract Akt/mTOR signaling influences CD4+ T cell differentiation; low levels favor Treg induction and high levels favor Th induction. Here, we identify several key elements in this pathway that are differentially regulated by TCR signal strength, including Akt, mTORC2 and the lipid phosphatase PTEN. The control of PTEN is poorly studied in T cells. Here, we identify multiple mechanisms that regulate PTEN. During Th induction, PTEN function is suppressed via lower mRNA levels, lower protein levels and an increase in phosphorylation. During Treg induction, PTEN function is maintained through the stabilization of PTEN mRNA transcription and sustained protein levels. Differential Akt/mTOR signaling regulates PTEN transcription via FoxO1. Additionally, Akt is controlled by the TCR signal. Phosphorylation of FoxO1 by Akt requires that Akt is phosphorylated on Thr308 and Ser473 by PDK1 and mTORC2 respectively. Different TCR signal strengths resulted in alternate phosphorylation on Akt where low TCR signal strength only induces phosphorylation of Thr308 on Akt. Low TCR signals result in the phosphorylation of an inhibitory site on Rictor, a subunit of mTORC2, thus providing a mechanism by which Akt activity is controlled by TCR signal strength. Proteomic data suggests that low and high TCR signal strength induce non-overlapping sets of phosphorylated Akt substrates. This work reveals how differential regulation of the Akt/mTOR pathway drives alternate CD4+ T cell fate outcomes.