PTEN does not modulate GLUT4 translocation in rat adipose cells under physiological conditions.

PTEN is a 3'-inositol lipid phosphatase that dephosphorylates products of PI 3-kinase. Since PI 3-kinase is required for many metabolic actions of insulin, we investigated the role of PTEN in insulin-stimulated translocation of GLUT4. In control rat adipose cells, we observed a approximately 2-fold increase in cell surface GLUT4 upon maximal insulin stimulation. Overexpression of wild-type PTEN abolished this response to insulin. Translocation of GLUT4 in cells overexpressing PTEN mutants without lipid phosphatase activity was similar to that observed in control cells. Overexpression of PTEN-CBR3 (mutant with disrupted membrane association domain) partially impaired translocation of GLUT4. In Cos-7 cells, overexpression of wild-type PTEN had no effect on ERK2 phosphorylation in response to acute insulin stimulation. However, Elk-1 phosphorylation in response to chronic insulin treatment was significantly decreased. Thus, when PTEN is overexpressed, both its lipid phosphatase activity and subcellular localization play a role in antagonizing metabolic actions of insulin that are dependent on PI 3-kinase but independent of MAP kinase. However, because translocation of GLUT4 in cells overexpressing a dominant inhibitory PTEN mutant (C124S) was similar to that of control cells, we conclude that endogenous PTEN may not modulate metabolic functions of insulin under normal physiological conditions.     

Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent Pathways cooperate to maintain lung cancer cell survival

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.     

Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics

Autophagy is a pathway in eukaryotes by which nutrient remobilization occurs through bulk protein and organelle turnover. Autophagy not only aides cells in coping with harsh environments but also plays a key role in many physiological processes that include pollen germination and tube growth. Most autophagic components are conserved among eukaryotes, but phylum-specific molecular components also exist. We show here that Arabidopsis thaliana PTEN, a protein and lipid dual phosphatase homologous to animal PTENs (phosphatase and tensin homologs deleted on chromosome 10), regulates autophagy in pollen tubes by disrupting the dynamics of phosphatidylinositol 3-phosphate (PI3P). The pollen-specific PTEN bound PI3P in vitro and was localized at PI3P-positive vesicles. Overexpression of PTEN caused accumulation of autophagic bodies and resulted in gametophytic male sterility. Such an overexpression effect was dependent upon its lipid phosphatase activity and was inhibited by exogenous PI3P or by expression of a class III phosphatidylinositol 3-kinase (PI3K) that produced PI3P. Overexpression of PTEN disrupted the dynamics of autophagosomes and a subpopulation of endosomes, as shown by altered localization patterns of respective fluorescent markers. Treatment with wortmannin, an inhibitor of class III PI3K, mimicked the effects by PTEN overexpression, which implied a critical role for PI3P dynamics in these processes. Despite sharing evolutionarily conserved catalytic domains, plant PTENs contain regulatory sequences that are distinct from those of animal PTENs, which might underlie their differing membrane association and thereby function. Our results show that PTEN regulates autophagy through phylum-specific molecular mechanisms.     

Hypothalamic glycogen synthase kinase 3β has a central role in the regulation of food intake and glucose metabolism

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lepob/ob mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.     

PtdIns(3,4,5)P(3)-dependent and -independent roles for PTEN in the control of cell migration

BACKGROUND: Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P(3) lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. RESULTS: We have investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. CONCLUSIONS: PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity.     

Substrate specificity and acute regulation of the tumour suppressor phosphatase, PTEN

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that functions as a PtdIns(3,4,5)P3 3-phosphatase to inhibit cell proliferation, survival and growth by antagonizing PI3K (phosphoinositide 3-kinase)-dependent signalling. Recent work has begun to focus attention on potential biological functions of the protein phosphatase activity of PTEN and on the possibility that some of its functions are phosphatase-independent. We discuss here the structural and regulatory mechanisms that account for the remarkable specificity of PTEN with respect to its PtdIns substrates and how it avoids the soluble headgroups of PtdIns that occur commonly in cells. Secondly we discuss the concept of PTEN as a constitutively active enzyme that is subject to negative regulation both physiologically and pathologically. Thirdly, we review the evidence that PTEN functions as a dual specificity phosphatase with discrete lipid and protein substrates. Lastly we present a current model of how PTEN may participate in the control of cell migration.     

Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons

To investigate whether phosphatidylinositol-3 kinase (PI3K) signaling mediates the metabolic effects of hypothalamic leptin action, adenoviral gene therapy was used to direct expression of leptin receptors to the area of the hypothalamic arcuate nucleus (ARC). This intervention markedly improved insulin sensitivity in genetically obese, leptin-receptor-deficient Koletsky (fa^k/fa^k) rats via a mechanism that was not dependent on reduced food intake but was attenuated by 44% by third-ventricular infusion of the PI3K inhibitor LY294002. Conversely, ARC-directed expression of a constitutively active mutant of protein kinase B (PKB/Akt, an enzyme activated by PI3K) mimicked the insulin-sensitizing effect of restored hypothalamic leptin signaling in these animals, despite having no effect on food intake or body weight. These findings suggest that hypothalamic leptin signaling is an important determinant of glucose metabolism and that the underlying neuronal mechanism involves PI3K.     

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.     

Phosphorylation of the survival kinase Akt by superoxide is dependent on an ascorbate-reversible oxidation of PTEN

In this report, we demonstrate that in serum-deprived mouse embryonic fibroblasts an increase in intracellular level of superoxide through pharmacological inhibition of the Cu/ZnSOD protein or the down-regulation of its expression using specific siRNA mimics growth factor-induced phosphorylation of Akt. Using the PI3K inhibitor LY294002 and PTEN knockout mouse embryonic fibroblasts, we show that phosphorylation of Akt by superoxide requires the production of PIP3 and that the target for the induction of Akt phosphorylation by O2.- is the phosphatase PTEN. Interestingly, the inhibition of PTEN involves an O2.--mediated oxidation of the phosphatase rather than regulation of its phosphorylation or decreased protein expression. Moreover, using differential reduction of oxidized protein by DTT and ascorbate, O2.--dependent oxidation of PTEN is shown to be due to S-nitrosylation of the protein. Finally, exposure of serum-deprived mouse embryonic fibroblasts to fetal bovine serum leads to a rapid and strong phosphorylation of Akt that is dependent on an ascorbate-reversible O2.--mediated oxidation of PTEN. These results support O2.- as a physiologically relevant second messenger for Akt activation through S-nitrosylation of PTEN and offer a mechanistic explanation for the mitogenic and prosurvival activities of O2.-.     

Protein kinase CK2/PTEN pathway plays a key role in platelet-activating factor-mediated murine anaphylactic shock

Platelet-activating factor (PAF) is a major mediator in the induction of fatal hypovolemic shock in murine anaphylaxis. This PAF-mediated effect has been reported to be associated with PI3K/Akt-dependent eNOS-derived NO. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is phosphatidylinositol phosphate phosphatase, which negatively controls PI3K by dephosphorylating the signaling lipid, phosphatidylinositol 3,4,5-triphosphate. In this study, we examined the possible involvement of PTEN in PAF-mediated anaphylactic shock. Induction of anaphylaxis or PAF injection resulted in a rapid decrease in PTEN activity, followed by increases in PI3K activity and phosphorylation of Akt and eNOS. Systemic administration of adenoviruses carrying PTEN cDNA (adenoviral PTEN), but not the control AdLacZ, not only attenuated anaphylactic symptoms, but also reversed anaphylaxis- or PAF-induced changes in PTEN and PI3K activities, as well as phosphorylation of Akt and eNOS. We found that the decreased PTEN activity was associated with PTEN phosphorylation, the latter effect being prevented by the protein kinase CK2 inhibitor, DMAT. DMAT also inhibited anaphylactic symptoms as well as the anaphylaxis- or PAF-mediated PTEN/PI3K/Akt/eNOS signaling cascade. CK2 activity was increased by PAF. The present data provide, as the key mechanism underlying anaphylactic shock, PAF triggers the upstream pathway CK2/PTEN, which ultimately leads to the activation of PI3K/Akt/eNOS. Therefore, CK2/PTEN may be a potent target in the control of anaphylaxis and other many PAF-mediated pathologic conditions.     

The significance of PTEN's protein phosphatase activity

Many hundreds of research papers over the last ten years have established the significance of PTEN's lipid phosphatase activity in mediating many of its effects on specific cellular processes in many different cell types, including cell growth, proliferation, survival, and migration ([Backman et al., 2002], [Iijima et al., 2002], [Leslie and Downes, 2002] and [Salmena et al., 2008]). In some cases, detailed signalling mechanisms have been identified by which these PtdInsP₃-dependent effects are manifest ([Kolsch et al., 2008], [Manning and Cantley, 2007] and [Tee and Blenis, 2005]). Further, in some settings, in vivo data from, for example genetic deletion of PTEN, relates closely with independent manipulation of the PI3K/Akt signalling pathway ([Bayascas et al., 2005], [Chen et al., 2006], [Crackower et al., 2002] and [Ma et al., 2005]). Together these studies indicate that the dominant effects of PTEN function are mediated through its regulation of PtdInsP₃-dependent signalling, but that its protein phosphatase activity also contributes in some settings. These conclusions are of great importance given the intense efforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experiments reviewed here have firmly established that the protein phosphatase activity of PTEN plays a role in the regulation of cellular processes including migration. On the other hand, it has not been established beyond doubt that PTEN acts on substrates other than itself; no such substrates have been confidently identified and effector mechanisms for PTEN's protein phosphatase activity are currently unclear. The goal for future research must be firstly to understand the signalling mechanisms by which PTEN protein phosphatase activity acts: whether this is through identifying substrates, or working out how autodephosphorylation mediates its effects. Secondly, and critically, the significance of PTEN's protein phosphatase activity must be established in vivo. This can be achieved through relating the phenotypes intervening with both PTEN and with protein phosphatase effector pathways when they are identified, and through the generation of mouse models expressing substrate selective PTEN mutants. We should then be able to answer the important question of whether PTEN's protein phosphatase activity contributes to tumour suppression.     

Redox regulation of PI 3-kinase signalling via inactivation of PTEN

The tumour suppressor PTEN is a PtdIns(3,4,5)P(3) phosphatase that regulates many cellular processes through direct antagonism of PI 3-kinase signalling. Here we show that oxidative stress activates PI 3-kinase-dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P(3) levels and activation of the downstream PtdIns(3,4,5)P(3) target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant-dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P(3) and oxidants are produced concurrently.     

Both p110α and p110β isoforms of PI3K can modulate the impact of loss-of-function of the PTEN tumour suppressor

The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN+/? mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P3, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN+/? mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P3 levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P3 levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.     

Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.     

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.     

Mechanisms of high-glucose/insulin-mediated desensitization of acute insulin-stimulated glucose transport and Akt activation

High-glucose/low-dose insulin-mediated insulin resistance of glucose transport was studied in 3T3-L1 adipocytes. In this model, proximal insulin signaling, including insulin receptor substrate (IRS)-1-bound phosphatidylinositol 3-kinase (PI 3-kinase) activation, is preserved, but insulin-stimulated protein kinase B (Akt) activation is markedly impaired. To assess a difference in acute insulin-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], cells were labeled with [32P]orthophosphate, and glycerophosphoinositides were quantified by HPLC. Although basal PtdIns(3,4,5)P3 was similar, insulin stimulated its production 33.6% more in controls (P < 0.03) than in insulin-resistant cells. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein, a lipid phosphatase that dephosphorylates PtdIns(3,4,5)P3 in the 3-position, was significantly and specifically increased in insulin-resistant cells. Treatment with rapamycin [a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1)] inhibited the increased PTEN expression and partially restored insulin-stimulated glucose transport and Akt activation to insulin-resistant cells. Acute insulin markedly stimulated Ser(636/639) phosphorylation of IRS-1; this was rapamycin inhibited but was significantly decreased in cells that had been preexposed to insulin, whereas total IRS-1 was unaffected. These findings were essentially paralleled by changes in the activation of p70 S6 kinase and S6-ribosomal protein. Overexpression of uncoupling protein-1 or manganese superoxide dismutase did not prevent the development of insulin-resistant glucose transport and impaired Akt activation in high-glucose/low-insulin-pretreated cells. The insulin resistance associated with glucotoxicity in our model reflects in part decreased availability of PtdIns(3,4,5)P3, which correlates with increased PTEN protein expression. Chronic activation of mTORC1 plays a role in stimulating PTEN expression and possibly in activation or induction of a phosphoprotein phosphatase. No evidence was found for a role for increased mitochondrial superoxide production in this model.