Protein Phosphatase Activity Of PTEN Research Articles

Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

The IGF1 signal pathway is highly activated in some subtype of gastric cancer(GC) that exhibits poor survival and chemotherapy resistance. Although the results of clinical trials of anti-IGF1R monoclonal antibodies and IGF-1R inhibitors have been mostly disappointing in unselected cancer patients, some patients benefit from anti-IGF1R therapy in these failed studies. Therefore, it is necessary to characterize the complex IGF signaling in GC and help refine the strategies targeting the IGF1 pathway. We found that GC cell lines exhibit differential responses to the specific IGF1R inhibitor OSI906. According to the phosphorylation status of Akt upon the OSI906 treatment, we divided the GC cell lines into IGF1R-dependent and IGF1R-independent cells. Both in vitro and in vivo experiments indicate that Dox-induced knockdown of NEDD4 significantly suppresses tumor growth of IGF1R-dependent GC cells and NEDD4 overexpression promotes tumor growth of IGF1R-dependent GC cells. In contrast, the proliferation of IGF1R-independent GC cells is not affected by NEDD4 silencing and overexpression. The rescue experiments show that a PTEN-IRS1 axis is required for NEDD4-mediated regulation of Akt activation and tumor growth in GC cells. Clinically, NEDD4 is expressed higher in IGF1-high GC tissues compared with IGF1-low GC tissues and normal tissues, and the co-high expression of NEDD4 and IGF1 predicts a worse prognosis in GC patients. Taken together, our study demonstrated that NEDD4 specifically promotes proliferation of GC cells dependent on IGF1/IGF1R signaling by antagonizing the protein phosphatase activity of PTEN to IRS1, and targeting NEDD4 may be a promising therapeutic strategy for IGF1 signal pathway-driven gastric cancer.

PTEN phosphatase inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6

PTEN encodes a tumor suppressor with lipid and protein phosphatase activities whose dysfunction has been implicated in melanomagenesis; less is known about how its phosphatases regulate melanoma metastasis. We demonstrate that PTEN expression negatively correlates with metastatic progression in human melanoma samples and a PTEN-deficient mouse melanoma model. Wildtype PTEN expression inhibited melanoma cell invasiveness and metastasis in a dose-dependent manner, behaviors that specifically required PTEN protein phosphatase activity. PTEN phosphatase activity regulated metastasis through Entpd5. Entpd5 knockdown reduced metastasis and IGF1R levels while promoting ER stress. In contrast, Entpd5 overexpression promoted metastasis and enhanced IGF1R levels while reducing ER stress. Moreover, Entpd5 expression was regulated by the ER stress sensor ATF6. Altogether, our data indicate that PTEN phosphatase activity inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6, thereby identifying novel candidate therapeutic targets for the treatment of PTEN mutant melanoma.

PTEN Protein Phosphatase Activity Is Not Required for Tumour Suppression in the Mouse Prostate.

Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or PtenY138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.

SHIP2 inhibition alters redox-induced PI3K/AKT and MAP kinase pathways via PTEN over-activation in cervical cancer cells.

Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) is required for protein kinase B (AKT) activation. The level of PI(3,4,5)P3 is constantly regulated through balanced synthesis by phosphoinositide 3-kinase (PI3K) and degradation by phosphoinositide phosphatases phosphatase and tensin homologue (PTEN) and SH2-domain containing phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 2 (SHIP2), known as negative regulators of AKT. Here, I show that SHIP2 inhibition in cervical cancer cell lines alters H2 O2 -mediated AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway activation. In addition, SHIP2 inhibition enhances reactive oxygen species generation. Interestingly, I found that SHIP2 inhibition and H2 O2 treatment enhance lipid and protein phosphatase activity of PTEN. Pharmacological targeting or RNA interference(RNAi) mediated knockdown of PTEN rescues extracellular signal-regulated kinase and AKT activation. Using a series of pharmacological and biochemical approaches, I provide evidence that crosstalk between SHIP2 and PTEN occurs upon an increase in oxidative stress to modulate the activity of mitogen-activated protein kinase and phosphoinositide 3/ATK pathways.

Control of Glucocorticoid Receptor Levels by PTEN Establishes a Failsafe Mechanism for Tumor Suppression

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.

PTEN activation contributes to neuronal and synaptic engulfment by microglia in tauopathy

Phosphatase and tensin homolog (PTEN) regulates synaptic density in development; however, whether PTEN also regulates synapse loss in a neurodegenerative disorder such as frontotemporal lobar degeneration with Tau deposition (FTLD-Tau) has not been explored. Here, we found that pathological Tau promotes early activation of PTEN, which precedes apoptotic caspase-3 cleavage in the rTg4510 mouse model of FTLD-Tau. We further demonstrate increased synaptic and neuronal exposure of the apoptotic signal phosphatidylserine that tags neuronal structures for microglial uptake, thereby linking PTEN activation to synaptic and neuronal structure elimination. By applying pharmacological inhibition of PTEN's protein phosphatase activity, we observed that microglial uptake can be decreased in Tau transgenic mice. Finally, we reveal a dichotomous relationship between PTEN activation and age in FTLD-Tau patients and healthy controls. Together, our findings suggest that in tauopathy, PTEN has a role in the synaptotoxicity of pathological Tau and promotes microglial removal of affected neuronal structures.

Ellagic acid, extracted from Sanguisorba officinalis, induces G1 arrest by modulating PTEN activity in B16F10 melanoma cells.

In Chinese medicine, herbal medicine is commonly used to treat individuals suffering from many types of diseases. We thus expected that some herbal medicines would contain promising compounds for cancer chemotherapy. Indeed, we found that Sanguisorba officinalis extracts strongly inhibit the growth of B16F10 melanoma cells, and we identified ellagic acid (EA) as the responsible ingredient. B16F10 cells treated with EA exhibited strong G1 arrest accompanied by accumulation of p53, followed by inactivation of AKT. Addition of a PTEN inhibitor, but not a p53 inhibitor, abrogated the EA-induced AKT inactivation and G1 arrest. The PTEN inhibitor also diminished EA-induced p53 accumulation. Furthermore, EA apparently increased the protein phosphatase activity of PTEN, as demonstrated by the reduced phosphorylation level of FAK, a protein substrate of PTEN. Furthermore, an in vitro PTEN phosphatase assay on PIP3 showed the direct modulation of PTEN activity by EA. These results suggest that EA functions as an allosteric modulator of PTEN, enhancing its protein phosphatase activity while inhibiting its lipid phosphatase activity. It is notable that a combination of EA and cisplatin, a widely used chemotherapy agent, dramatically enhanced cell death in B16F10 cells, suggesting a promising strategy in chemotherapy.

PTEN Suppresses Glycolysis by Dephosphorylating and Inhibiting Autophosphorylated PGK1

The PTEN tumor suppressor is frequently mutated or deleted in cancer and regulates glucose metabolism through the PI3K-AKT pathway. However, whether PTEN directly regulates glycolysis in tumor cells is unclear. We demonstrate here that PTEN directly interacts with phosphoglycerate kinase 1 (PGK1). PGK1 functions not only as a glycolytic enzyme but also as a protein kinase intermolecularly autophosphorylating itself at Y324 for activation. The protein phosphatase activity of PTEN dephosphorylates and inhibits autophosphorylated PGK1, thereby inhibiting glycolysis, ATP production, and brain tumor cell proliferation. In addition, knockin expression of a PGK1 Y324F mutant inhibits brain tumor formation. Analyses of human glioblastoma specimens reveals that PGK1 Y324 phosphorylation levels inversely correlate with PTEN expression status and are positively associated with poor prognosis in glioblastoma patients. This work highlights the instrumental role of PGK1 autophosphorylation in its activation and PTEN protein phosphatase activity in governing glycolysis and tumorigenesis.

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.

The role of PTEN - HCV core interaction in hepatitis C virus replication

Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid phosphatase activity. PTEN has no effect on HCV RNA translation. PTEN decreases HCV replication and the protein phosphatase activity of PTEN is essential for this function. PTEN interacts with the HCV core protein and requires R50 in domain I of HCV core and PTEN residues 1–185 for this interaction. This interaction is required for PTEN-mediated inhibition of HCV replication. This gives rise to a reduction in PTEN levels and intracellular lipid abundance, which may in turn regulate HCV replication. HCV core domain I protein increases the lipid phosphatase activity of PTEN in an in vitro assay, suggesting that HCV infection can also regulate PTEN. Taken together, our results demonstrated an important regulatory role of PTEN in the HCV life cycle.

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.

Direct regulation of transforming growth factor β-induced epithelial-mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells.

Transforming growth factor β (TGFβ) causes the acquisition of epithelial-mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ-induced phosphorylation of its C-terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C-terminus (PTEN4A), but not PTEN wild, inhibits TGFβ-induced EMT. Nevertheless, whether or not the blockade of TGFβ-induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C-terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ-induced EMT in lung cancer cells. The unphosphorylated PTEN C-terminus might not directly retain the phosphatase activities and repress TGFβ-induced EMT; the modification that keeps the PTEN C-terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ-induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C-terminus might be a therapeutic target to negatively regulate TGFβ-induced EMT in lung cancer cells.

Abstract IA10: Nuclear PTEN in regulation of genome integrity

Abstract Loss of PTEN function has been implicated in the etiology of numerous human malignancies, including sporadic brain, prostate, thyroid, breast, uterine and kidney tumors. PTEN germline have also been found in patients with PHTS, an autosomal-dominant cancer predisposition syndrome. Despite a thorough understanding of the inhibitory role for PTEN in the PI3K pathway, acting as a 3′PI phosphatase and directly antagonizing PI3'K, relatively little is known about PTEN's functions in the nucleus, a cellular compartment where it also resides. Our recent work has uncovered a nuclear function of PTEN in tumor suppression, which is independent from its 3′PI-phosphatase activity, but relies on PTEN protein phosphatase activity. Nuclear PTEN, which is SUMOylated, plays a prominent role in homologous recombination-based DNA repair, acting downstream of the protein kinase ATM. PTEN-deficient cells and tumors are impaired in their response to DNA damage and display heightened sensitivity to genotoxic agents, which may have significant implications for the treatment of PTEN-deficient cancers. Citation Format: Vuk Stambolic. Nuclear PTEN in regulation of genome integrity. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA10.

The protein phosphatase activity of PTEN is essential for regulating neural stem cell differentiation.

BackgroundThe tumor suppressor gene Phosphatase and tensin homolog (PTEN) is highly expressed in neural progenitor cells (NPCs) and plays an important role in development of the central nervous system. As a dual-specificity phosphatase, the loss of PTEN phosphatase activity has been linked to various diseases.ResultsHere we report that the protein phosphatase activity of Pten is critical for regulating differentiation of neural progenitor cells. First we found that deletion of Pten promotes neuronal differentiation. To determine whether the protein or lipid phosphatase activity is required for regulating neuronal differentiation, we generated phosphatase domain-specific Pten mutations. Interestingly, only expression of protein phosphatase-deficient mutant Y138L could mimic the effect of knocking down Pten, suggesting the protein phosphatase of Pten is critical for regulating NPC differentiation. Importantly, we showed that the wild-type and lipid phosphatase mutant (G129E) forms of Pten are able to rescue neuronal differentiation in Pten knockout NPCs, but mutants containing protein phosphatase mutant cannot. We further found that Pten-dependent dephosphorylation of CREB is critical for neuronal differentiation.ConclusionOur data indicate that the protein phosphatase activity of PTEN is critical for regulating differentiation of NSCs during cortical development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0114-1) contains supplementary material, which is available to authorized users.

Pnck overexpression in HER-2 gene-amplified breast cancer causes Trastuzumab resistance through a paradoxical PTEN-mediated process.

The gene for Pregnancy Up-regulated Non-ubiquitous Calmodulin Kinase (Pnck), a novel calmodulin kinase, is expressed in roughly one-third of human breast tumors, but not in adjoining normal tissues. Pnck alters EGFR stability and function, prompting this study to determine if Pnck expression has implications for HER-2 function and HER-2-directed therapy. The frequency of Pnck expression in HER-2-amplified breast cancer was examined by immunohistochemistry, and the impact of Pnck expression in the presence of HER-2 amplification on cancer cell proliferation, clonogenicity, cell-cycle progression, and Trastuzumab sensitivity was examined in vitro by transfection of cells with Pnck. Cell signaling was probed by Western blot analysis and shRNA-mediated PTEN knockdown. Over 30 % of HER-2 amplified tumors were found to express Pnck. Expression of Pnck in SkBr3 cells resulted in increased proliferation, clonal growth, cell-cycle progression, and Trastuzumab resistance. Pnck expression increases Hsp27 expression, Trastuzumab partial agonist activity on HER-2 Y1248 phosphorylation, and suppressed extracellular signal-regulated kinase (ERK1/2) activity. Knockdown of endogenous PTEN upregulated ERK1/2 activity, inhibited cellular proliferation, and partially sensitized Pnck/SKBr3 cells to Trastuzumab treatment. Increased proliferation of the Pnck/SKBr3 cells was observed following expression of protein phosphatase active and lipid phosphatase dead PTEN mutant but not the total phosphatase dead PTEN mutant. Co-overexpression of HER-2 and Pnck results in enhanced tumor cell proliferation and Trastuzumab resistance that is paradoxically dependent on PTEN protein phosphatase activity. This suggests that Pnck may be a marker of Trastuzumab resistance and possibly a therapeutic target.

PTEN, here, there, everywhere

PTEN, here, there, everywhere

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.

Focal Adhesion Kinase Inactivation Reduces the Development of Acute Leukemia and Partially Rescues Hematopoietic Stem Cell Defects in Pten-Knockout Mice

AbstractAbstract 864Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten-knockout in adult mice (Pten−/−) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten−/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten−/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten’s lipid phosphatase activity. However, the role of Pten’s protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied.Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre+Ptenfl/flFakfl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten−/−Fak−/−) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten−/−Fak−/− mice is significantly reduced compared to Pten−/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten−/−Fak−/− mice compared to Pten−/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten−/−Fak−/− HSCs is significantly improved compared to Pten−/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten−/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten−/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten−/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. 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.

Functional analysis of the protein phosphatase activity of PTEN

In vitro, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) displays intrinsic phosphatase activity towards both protein and lipid substrates. In vivo, the lipid phosphatase activity of PTEN, through which it dephosphorylates the 3 position in the inositol sugar of phosphatidylinositol derivatives, is important for its tumour suppressor function; however, the significance of its protein phosphatase activity remains unclear. Using two-photon laser-scanning microscopy and biolistic gene delivery of GFP (green fluorescent protein)-tagged constructs into organotypic hippocampal slice cultures, we have developed an assay of PTEN function in living tissue. Using this bioassay, we have demonstrated that overexpression of wild-type PTEN led to a decrease in spine density in neurons. Furthermore, it was the protein phosphatase activity, but not the lipid phosphatase activity, of PTEN that was essential for this effect. The ability of PTEN to decrease neuronal spine density depended upon the phosphorylation status of serine and threonine residues in its C-terminal segment and the integrity of the C-terminal PDZ-binding motif. The present study reveals a new aspect of the function of this important tumour suppressor and suggest that, in addition to dephosphorylating the 3 position in phosphatidylinositol phospholipids, the critical protein substrate of PTEN may be PTEN itself.