PTEN Lipid Research Articles

Rapid estrogen signaling negatively regulates PTEN activity through phosphorylation in endometrial cancer cells.

Hyperestrogenicity is a risk factor for endometrial cancer. 17β-estradiol (E2) is known to stimulate both genomic and nongenomic estrogen receptor-α (ERα) actions in a number of reproductive tissues. However, the contributions of transcription-independent ERα signaling on normal and malignant endometrium are not fully understood. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that decreases cellular mitosis primarily through negative regulation of the phosphoinositide 3-kinase/AKT signaling axis. PTEN levels are elevated during the E2 dominated, mitotically active, proliferative phase of the menstrual cycle, indicating possible hormonal regulation of PTEN in the uterus. In order to determine if rapid E2 signaling regulates PTEN, we used ERα-positive, PTEN-positive, endometrial cells. We show that cytosolic E2/ERα signaling leads to increased phosphorylation of PTEN at key regulatory residues. Importantly, E2 stimulation decreased PTEN lipid phosphatase activity and caused consequent increases in phospho-AKT. We further demonstrate that cytosolic ERα forms a complex with PTEN in an E2-dependent manner, and that ERα constitutively complexes with protein kinase2-α (CK2α), a kinase previously shown to phosphorylate the C-terminal tail of PTEN. These results provide mechanistic support for an E2-dependent, ERα cytosolic signaling complex that negatively regulates PTEN activity through carboxy terminus phosphorylation. Using an animal model, we show that sustained E2 signaling results in increased phospho-PTEN (S380, T382, and T383), total PTEN, and phospho-AKT (S473). Taken together, we provide a novel mechanism in which transcription-independent E2/ERα signaling may promote a pro-tumorigenic environment in the endometrium.

Adult B-cell acute lymphoblastic leukemia cells display decreased PTEN activity and constitutive hyperactivation of PI3K/Akt pathway despite high PTEN protein levels

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

The Dynamics of P-Rex 2 Membrane and Protein Interactions

P-Rex 2 (phosphatidylinositol (3,4,5)-trisphosphate Rac exchanger 2) protein is a Rac guanine nucleotide exchange factor (GEF) whose activity is tightly regulated through activation by PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) and G-protein βγ subunits along with phosphorylation and domain-domain interactions. A previous study has shown that P-Rex 2 DH-PH domains interacts with PTEN (phosphate and tensin homologue deleted on chromosome 10), which is a frequently mutated tumor suppressor in human cancer that dephosphorylates PI(3,4,5)P3 and antagonizes PI3K signaling. P-Rex 2 binds to PTEN through its DH-PH domain, inhibits PTEN's lipid phosphatase activity and activates the PI3K pathway. This suggests that P-Rex 2 may function as a regulator of PTEN and activator of the PI3K pathway, thereby, contributing to a variety of pathological and physiological processes, such as tumorigenesis, diabetes, and aging. We are investigating the role of P-Rex 2 DH-PH domains in lipid binding and PTEN protein binding using steady-state fluorescence, stopped flow photometry, and ITC (isothermal titration calorimetry). This study aims to delineate P-Rex 2 membrane and protein interactions at the molecular level.

PTEN, here, there, everywhere

PTEN, here, there, everywhere

Characterization of a Tumor-Associated Activating Mutation of the p110β PI 3-Kinase

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β.

PTEN is involved in modulation of vasculogenesis in early chick embryos

SummaryPTEN is a tumor suppressor gene that is mutated and/or deleted in many types of tumor. This gene also plays a very distinct role in the early stages of embryonic development such as cell migration, proliferation and migration. Nevertheless, little is known of the function of PTEN in vasculogenesis during chick embryonic development. In this study, we used in situ hybridization to first demonstrate the expression pattern of PTEN during gastrulation. PTEN was found mainly expressed in the blood islands of area opaca, the neural tube and mesodermal structures. Overexpression of PTEN obstructed the epithelial–mesenchymal transition (EMT) process in the primitive streak. EMT is the first prerequisite required for the emigration of hemangioblasts during vasculogenesis. When PTEN expression was silenced, we observed that it produced an adverse effect on mesodermal cell emigration to the extra-embryonic blood islands. In addition, we also demonstrated that even if the perturbed-PTEN cells did not affect the formation of blood islands, migrant mesodermal cells overexpressing wt PTEN-GFP had difficulties integrating into the blood islands. Instead, these cells were either localized on the periphery of the blood islands or induced to differentiate into endothelial cells if they managed to integrate into blood islands. Development of the intra-embryonic primary vascular plexus was also affected by overexpression of PTEN. We proposed that it was elevated PTEN lipid phosphatase activity that was responsible for the morphogenetic defects induced by PTEN overexpression. In this context, we did not find PTEN affecting VEGF signaling. In sum, our study has provided evidence that PTEN is involved in vasculogenesis during the early stages of chick embryo development.

PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism

Hepatitis C virus (HCV) infection is dependent on lipid metabolism. Hepatocyte steatosis occurs frequently in HCV infection, but the relationship between steatosis and HCV life cycle is unclear. We showed that HCV induces steatosis via the downregulation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). We here investigated how PTEN may affect HCV production. The effect of overexpression or silencing of PTEN on HCV secretion was assessed in genomic-length Jc1 infected HuH7 cells. The role of PTEN protein and lipid phosphatase activities on lipid metabolism and infectious viral particle secretion was investigated using dominant-negative PTEN mutants. The importance of cholesterol metabolism for PTEN-dependent lipid droplet biogenesis and viral particle secretion was examined using statins. PTEN silencing in Jc1 infected HuH7 cells stimulated HCV particle secretion, while PTEN overexpression decreased virus egress. Viral secretion was also increased by overexpression of protein phosphatase-deleted (PTENY138L), but not lipid phosphatase-deleted (PTENG129E), PTEN mutant, thus indicating that the protein phosphatase activity of PTEN controls viral secretion. Similarly, PTENY138L, but not PTENG129E mutant induced the formation of large lipid droplets. PTENY138L mutant did not affect biosynthesis of triglycerides, but promoted the biosynthesis of cholesterol esters. Consistently, statins prevented the increased cholesterol ester production, large lipid droplet formation, and viral secretion in cells expressing the PTENY138L mutant. Downregulation of PTEN protein phosphatase activity by HCV affects cholesterol metabolism, thereby inducing the appearance of large lipid droplets and increasing virion egress.

PTEN-lipid Binding Assay

The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which they attach. Then the membrane is incubated with a lipid binding protein possessing an epitope tag. The protein binds to the lipid which is detected by immunoblotting with an antibody recognizing the epitope tag (see Figure 1). PTEN is an important tumor suppressor which functions as both protein and lipid phosphatase. The primary physiological substrate of PTEN is signaling lipid PtdIns (3, 4, 5) P3, by dephosphrylating PtdIns (3, 4, 5) P3 to PtdIns (4, 5) P2 PTEN negatively regulates PI3K signaling and mediates its tumor-suppressor function by inactivating downstream oncogenic AKT-mediated signaling. The PTEN lipid binding assay is conducted to study the specific binding of PTEN to different lipid molecules.

Pten Binding to Morphologically Heterogenous Membrane Environments

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor protein that dephosphorylates phosphoinositides at the 3-position of the inositol ring. PTEN's membrane association has been shown to be a complex process that involves binding to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylserine [PS]. Recent evidence suggests that the presence of cholesterol promotes the formation of PI(4,5)P2 enriched domains. This study has three objectives: First, to investigate PTEN's binding to lipid systems with laterally heterogeneous membrane morphology. Second, to delineate the kinetics of PTEN lipid binding and activation using stopped-flow fluorescence measurements. Third, by using single molecule fluorescence microscopy determine the lateral diffusion of membrane bound PTEN on supported bilayer and how the binding is affected by laterally heterogeneous membrane environments. Overall the study aims to understand the dynamics of PTEN-membrane interactions at the molecular level.

PD-1 Decreases PTEN Protein Stability While Increasing PTEN Phosphatase Activity by Inhibiting CK2.

Abstract 2145Maintenance of peripheral tolerance is essential for homeostasis of the immune system. The newer pathway of the B7:CD28 family consisting of the receptor PD-1 (CD279) and its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), plays a vital role in the induction and maintenance of T cell anergy and peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis but can also compromise anti-tumor and anti-viral immunity. The precise mechanisms via which PD-1 mediates its inhibitory effects in T cells remain elusive. One of the critical signaling pathways targeted by PD-1 is the PI3K/Akt pathway, via which PD-1 mediates inhibitory effects on T cell proliferation, cytokine production and glucose uptake. In contrast to CTLA-4, which affects PI3/Akt signaling by targeting Akt through PP2A activation, PD-1 targets the PI3K/Akt pathway by directly inhibiting activation of PI3K. It has been proposed that PD-1 inhibits PI3K by upregulating PTEN (phosphatase and tensin homologue deleted on chromosome 10), a phosphoinositole 3,4,5-trisphosphatase important for antagonizing PI3K signaling, but the mechanism remains unclear. In the present study we sought to dissect the effects of PD-1 mediated signaling on the regulation of PTEN expression and function. We used highly purified primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies (mAbs) against CD3 and CD28 with or without agonist mAb against PD-1. As determined by assessment of PTEN lipid phosphatase activity, stimulation of T cells via TCR/CD3, CD28 and PD-1 resulted in prominent increase of PTEN phosphatase activity compared to T cells stimulated via TCR/CD3 and CD28 without PD-1 signals. Surprisingly, although T cells receiving PD-1 signals displayed increased PTEN phosphatase activity they expressed lower levels of PTEN compared to T cells stimulated via TCR/CD3 and CD28, as determined by both western blot and by flow cytometry. In other cell types, the abundance of PTEN can be regulated transcriptionally and through phosphorylation-dependent modulation of protein stability. Assessment of PTEN mRNA by real time qPCR indicated that PTEN mRNA levels were comparable between T cells stimulated via TCR/CD3/CD28 and T cells stimulated via TCR/CD3/CD28 and PD-1. PTEN possesses a C-terminal non-catalytic regulatory domain, which plays an important role in its biological activity. A cluster of serine/threonine residues within this region is phosphorylated by the serine/threonine kinase casein kinase 2 (CK2). CK2-mediated phosphorylation stabilizes PTEN by preventing its ubiquitin-dependent degradation while decreasing PTEN phosphatase activity. In contrast, impaired phosphorylation in this region results in decreased PTEN protein stability but increased phosphatase activity. Because PD-1 signaling resulted in reduced PTEN protein expression but increased phosphatase activity, we examined whether PD-1 might alter CK2 activity and CK2-mediated phosphorylation of PTEN. In CD4+ T cells stimulated via TCR/CD3 and CD28, CK2 activation was robust and resulted in sustained phosphorylation on PTEN S380/T382/383 cluster. In contrast, T cells receiving PD-1 signals displayed impaired CK2 activity and significantly diminished phosphorylation of PTEN in the S380/T382/383 cluster. To determine whether downregulation of PTEN protein expression via PD-1 was mediated via proteasome-dependent degradation we used the proteasome inhibitor MG132. Stimulation of T cells via TCR/CD3, CD28 and PD-1 in the presence of MG132 prevented PD-1-mediated decrease of PTEN and resulted in reduced PTEN activity. Our results identify CK2 as a new target of PD-1-mediated signaling and reveal an unexpected mechanism via which PD-1 regulates PTEN phosphatase activity and inhibition of the PI3K/Akt pathway. Disclosures:No relevant conflicts of interest to declare.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

PTEN Lipid Phosphatase Activity and Proper Subcellular Localization Are Necessary and Sufficient for Down-Regulating AKT Phosphorylation in the Nucleus in Cowden Syndrome

Germline mutations in PTEN are associated with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) hamartoma tumor syndrome including Cowden syndrome (CS) and Cowden-like syndrome (CSL) that predisposes to high risks of benign and malignant tumors of thyroid and breast. The objective of the study was to analyze the subcellular pattern of phosphorylated (P)-AKT expression in nonmedullary thyroid cancers from PTEN hamartoma tumor syndrome patients and to investigate whether the lack of PTEN in the nucleus and/or lack of proper PTEN function in the nucleus affect(s) nuclear AKT activity in CS patients. In all, 664 patients with CS/CSL were screened for PTEN germline mutations and nonmedullary thyroid cancers. Twenty-two patients who have both pathogenic PTEN germline mutations and nonmedullary thyroid cancers were selected. Thyroid samples from these patients were stained for PTEN and P-AKT. In our in vitro study, PTEN was knocked down or overexpressed in both thyroid cancer cells and breast cancer cells, and nuclear P-AKT was compared with the control. Loss of PTEN protein was found in thyroid adenomas and carcinomas from all 22 (100%) PTEN(Mut+) CS/CSL patients. AKT activation was identified in 17 of 22 (77.3%) thyroid adenoma/carcinoma specimens, and most patients (63.7%) have activated nuclear AKT. Knockdown of PTEN in cells containing wild-type PTEN enhanced nuclear P-AKT, whereas expression of wild-type PTEN, but not phosphatase-dead mutants (C124S or G129E), markedly reduced nuclear P-AKT in PTEN null cells. We also showed that in breast cancer but not thyroid cancer cells, PTEN suppresses nuclear P-AKT mainly through decreasing P-AKT nuclear translocation by reducing the PIP3/P-AKT reservoir in the cytoplasm. In thyroid cancer cells, PTEN suppresses phosphorylation of AKT already resident in the nucleus. PTEN is necessary and sufficient for inhibiting AKT activation in the nucleus through its intact lipid phosphatase activity and proper subcellular localization.

PTEN: a new player controlling structural and functional synaptic plasticity

Phosphoinositide 3-kinase (PI 3-kinase) is a ubiquitous enzyme that catalyses the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI 3-kinase driven generation of PIP3 activates various downstream signalling cascades which in turn regulate numerous biological processes including growth, metabolism, proliferation, migration and cell size. PTEN (phosphatase and tensin homologue deleted on chromosome 10), which was originally identified as a tumour promoter, is a constitutively active lipid phosphatase that opposes PI 3-kinase-dependent signalling by promoting dephosphorylation of PIP3 into PIP2. PTEN is highly expressed in neurons and several lines of evidence support a role for PTEN in regulating important neuronal functions. Indeed, familial mutations that result in PTEN inactivation have been linked to neurological disorders such as ataxia, mental retardation and seizures (Backman et al. 2001). Moreover, loss of PTEN function at early stages of development results in widespread deficits in neuronal growth, synaptogenesis and synaptic plasticity suggesting additional roles for PTEN in these processes. However, it is not clear if PTEN-linked synaptic impairments are a direct consequence of loss of PTEN function or are secondary to PTEN-driven alterations in neuronal morphology. A recent insightful study by Sperow et al. (2012) in The Journal of Physiology aims to address this issue. This study demonstrates that the structural and functional properties of hippocampal synapses are independently controlled by PTEN and that PTEN plays a direct role in activity-dependent hippocampal synaptic plasticity. Several studies have observed that a key feature of deleting PTEN during CNS development is progressive hypertrophy of neuronal dendrites and spines as well as a marked increase in the size of neurons and synapses. PTEN deletion or down-regulation in hippocampal neurons also results in impairments in two forms of activity-dependent synaptic plasticity, namely long-term potentiation (LTP) and long-term depression (LTD). In order to determine if the structural and functional effects of PTEN on hippocampal synapses are related, Sperow and colleagues generated transgenic mice with conditional knockout of PTEN in forebrain excitatory neurons. As PTEN deletion was apparent after postnatal day 14 (P14) in these mice, the impact of PTEN deletion on hippocampal synaptic function could be examined prior to its hypertrophic effects. Postnatal deletion of PTEN had no significant effect on excitatory synaptic transmission at hippocampal CA1 synapses, as basal synaptic transmission and paired pulse facilitation was normal in PTEN−/− mice compared to wild-type littermates. In contrast, deficits in both hippocampal LTP and LTD were observed in PTEN−/− mice. Postnatal deletion of PTEN also resulted in hippocampal-specific memory deficits as significant impairments in spatial memory tasks performed in the Morris water maze were observed in PTEN−/− mice. However, no differences in soma size or dendritic morphology were detected between PTEN+ and PTEN− neurons in PTEN−/− mice, suggesting that structural abnormalities do not contribute to the deficits in synaptic plasticity following postnatal PTEN deletion. As PTEN negatively regulates PI 3-kinase driven signalling, deletion or inhibition of PTEN results in uncontrolled stimulation of downstream components of the PI 3-kinase cascade, including the protein kinase PDK1. Consequently reducing PDK1 expression can reverse some neuronal abnormalities linked to PTEN deletion (Chalhoub et al. 2009). In this study, Sperow et al. examined whether postnatal deletion of PDK1 rescued the impairments in hippocampal synaptic plasticity in PTEN−/− mice. Postnatal deletion of PDK1 had no effect on hippocampal synaptic plasticity or neuronal morphology per se. However, deletion of PDK1 rescued the synaptic plasticity deficits, as the magnitude of hippocampal LTP and LTD was comparable in double mutant mice (PTEN−/−; PDK1−/−) and wild-type littermates. In contrast, however, significant impairments in spatial memory were still observed in the double mutant mice compared to wild-type littermates, suggesting that PDK1 deletion is able to counterbalance the effects of postnatal PTEN deletion on synaptic plasticity but not on spatial memory. In conclusion this study indicates that hippocampal synaptic plasticity and neuronal morphology are independently regulated by PTEN in vivo. It also highlights an important role for PTEN in hippocampal LTP and LTD. Although the precise cellular mechanisms underlying PTEN modulation of synaptic plasticity are not fully known, recent studies suggest involvement of a postsynaptic mechanism as PTEN inhibition promotes AMPA receptor trafficking to synapses leading to a persistent increase in excitatory synaptic strength in adult hippocampal slices (Moult et al. 2010). Conversely enhanced PTEN lipid phosphatase activity has been reported to depress excitatory synaptic transmission, which is in turn required for NMDA receptor-dependent LTD (Jurado et al. 2011). Together these findings provide important new insights into the role of PTEN in regulating hippocampal synaptic function.

Arresting times for PTEN

Arresting times for PTEN

Protein Interactions of Phosphatase and Tensin Homologue (PTEN) and Its Cancer-associated G20E Mutant Compared by Using Stable Isotope Labeling by Amino Acids in Cell Culture-based Parallel Affinity Purification

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-type-specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.

Abstract LB-132: Protein interactome between PTEN and cancer-associated mutant demonstrated the regulatory pathway of migration through its novel interactors

Abstract Mutation of the PTEN tumor suppressor protein frequently occurs in a variety of human cancers. PTEN is a phosphatase that negatively regulates the phosphatidyl-inositol-3-kinase (PI3K) pathway, and regulates cell proliferation, apoptosis, and cell motility. Though the tumor suppressive function involves PTEN's lipid phosphatase-dependent and -independent activities, the mechanism leading to the phosphatase-independent function of PTEN is poorly understood. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here we use a cancer associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using mass spectrometry (MS)-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach are mainly involved in cell migration and apoptosis pathways, suggesting deregulation of the pathways could be involved in the tumorigenesis derived by the PTEN mutant. We further demonstrate that the one of wild-type specific interactor is required for the regulatory function of wild-type PTEN in cell migration. Furthermore, the other wild type specific interactor affects cell growth. These finding contribute to a better understanding of the mechanisms of PTEN's functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-132. doi:10.1158/1538-7445.AM2011-LB-132

Abstract 1446: Dissecting the role of PTEN/Akts in metastatic melanoma

Abstract Melanoma is an aggressive human cancer whose incidence is increasing more rapidly than any other cancer in the United States. It is characterized by an extremely high potential to develop metastases and poor response to current therapy. Although the genetic studies indicated that melanoma genesis related with molecular lesions in c-MET, INK4a, Ras/Raf, p53 and PTEN pathways, the molecular mechanism inciting metastatic melanoma are poorly understood. PTEN is a major tumor suppressor in humans. To study the role of PTEN in progression of human melanoma, we first used tissue microarrays to examine PTEN expression in 30 lymph node melanoma metastases, 30 primary melanomas, and 9 normal skin tissues. Our results showed that PTEN loss was significantly correlated with progression to the metastatic state. Moreover, analysis of various human melanoma cell lines demonstrated that PTEN expression was related to metastatic behavior, supporting the notion that PTEN is involved in the metastatic regulation of melanoma. PTEN has phosphatase activity that can act upon both polypeptide and phosphoinositide substrates. The PTEN lipid phosphatase is a ubiquitous regulator of PI3K signaling pathway. The loss of expression or mutational inactivation of PTEN protein enhances the phosphorylation of PKB/AKT and thus influences cell survival signaling. Interestingly, a PTEN mutant, which loses lipid but not protein phosphatase activity, lacks the ability to induce either G1 arrest or apoptosis, but nonetheless retains the ability to inhibit cell spreading and motility, suggesting that PTEN protein phosphatase activity is important for these behaviors. To further dissect the role of this PTEN activity in metastasis, we have established a model system containing exploiting mutant PTEN proteins with various phosphatase activities. We found that wild type PTEN can inhibit cell motility and invasiveness, while PTEN mutants cannot. Notably, wild type PTEN was able to reduce the metastatic potential of melanoma cells, whereas phosphatase deficient PTEN could promote the melanoma cell metastatic behavior. We found that PTEN mediates metastatic activity through the AKT pathway. We further determined that Akt 3 was key mediators of metastatic potential in melanoma cells in vivo. Akt3 could also rescue PTEN-mediated inhibition of metastasis. PTEN loss and Akt3 overexpression functioned synergistically to promote metastasis. Our data demonstrate that metastatic behavior in melanoma cells is dependent on the status of PTEN protein phosphatase activity and downstream Akt3, and suggest that AKT/PTEN signaling should be considered as a therapeutic target in metastatic melanoma Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2011-1446

Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) Inhibition by 4-Hydroxynonenal Leads to Increased Akt Activation in Hepatocytes

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is proposed to be an important factor in the etiology of alcoholic liver disease. To understand the effects of 4-HNE on homeostatic signaling pathways in hepatocytes, cellular models consisting of the human hepatocellular carcinoma cell line (HepG2) and primary rat hepatocytes were evaluated. Treatment of both HepG2 cells and primary hepatocytes with subcytotoxic concentrations of 4-HNE resulted in the activation of Akt within 30 min as demonstrated by increased phosphorylation of residues Ser473 and Thr308. Quantification and subsequent immunocytochemistry of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)[rsqb] resulted in a 6-fold increase in total PtdIns(3,4,5)P(3) and increased immunostaining at the plasma membrane after 4-HNE treatment. Cotreatment of HepG2 cells with 4-HNE and the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) or the protein phosphatase 2A (PP2A) inhibitor okadaic acid revealed that the mechanism of activation of Akt is PI3K-dependent and PP2A-independent. Using biotin hydrazide detection, it was established that the incubation of HepG2 cells with 4-HNE resulted in increased carbonylation of the lipid phosphatase known as "phosphatase and tensin homolog deleted on chromosome 10" (PTEN), a key regulator of Akt activation. Activity assays both in HepG2 cells and recombinant PTEN revealed a decrease in PTEN lipid phosphatase activity after 4-HNE application. Mass spectral analysis of 4-HNE-treated recombinant PTEN detected a single 4-HNE adduct. Subsequent analysis of Akt dependent physiological consequences of 4-HNE in HepG2 cells revealed significant increases in the accumulation of neutral lipids. These results provide a potential mechanism of Akt activation and cellular consequences of 4-HNE in hepatocytes.

The ER UDPase ENTPD5 Promotes Protein N-Glycosylation, the Warburg Effect, and Proliferation in the PTEN Pathway

PI3K and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinases that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. We report here that ENTPD5, an endoplasmic reticulum (ER) enzyme, is upregulated in cell lines and primary human tumor samples with active AKT. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN null cells causes ER stress and loss of growth factor receptors. ENTPD5, together with cytidine monophosphate kinase-1 and adenylate kinase-1, constitute an ATP hydrolysis cycle that converts ATP to AMP, resulting in a compensatory increase in aerobic glycolysis known as the Warburg effect. The growth of PTEN null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an integral part of the PI3K/PTEN regulatory loop and a potential target for anticancer therapy.

PTEN is recruited to the postsynaptic terminal for NMDA receptor-dependent long-term depression

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important regulator of phosphatidylinositol-(3,4,5,)-trisphosphate signalling, which controls cell growth and differentiation. However, PTEN is also highly expressed in the adult brain, in which it can be found in dendritic spines in hippocampus and other brain regions. Here, we have investigated specific functions of PTEN in the regulation of synaptic function in excitatory hippocampal synapses. We found that NMDA receptor activation triggers a PDZ-dependent association between PTEN and the synaptic scaffolding molecule PSD-95. This association is accompanied by PTEN localization at the postsynaptic density and anchoring within the spine. On the other hand, enhancement of PTEN lipid phosphatase activity is able to drive depression of AMPA receptor-mediated synaptic responses. This activity is specifically required for NMDA receptor-dependent long-term depression (LTD), but not for LTP or metabotropic glutamate receptor-dependent LTD. Therefore, these results reveal PTEN as a regulated signalling molecule at the synapse, which is recruited to the postsynaptic membrane upon NMDA receptor activation, and is required for the modulation of synaptic activity during plasticity.