Ubiquitin Ligase β-TrCP Research Articles

Xom, a ventralizing factor, regulates beta-catenin levels and cell fate.

Read the Original article at doi:10.1002/1873-3468.12949

S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer.

Rationale: Mxi1 is regarded as a potential tumor suppressor protein that antagonizes the transcriptional activity of proto-oncogene Myc. However, the clinical significances and underlying mechanisms by which Mxi1 is regulated in lung cancer remain poorly understood.Methods: Mass spectrometry analysis and immunoprecipitation assay were utilized to detect the protein-protein interaction. The phosphorylation of Mxi1 was evaluated by in vitro kinase assays. Poly-ubiquitination of Mxi1 was examined by in vivo ubiquitination assay. Lung cancer cells stably expressing wild-type Mxi1 or Mxi1-S160A were used for functional analyses. The expression levels of Mxi1 and S6K1 were determined by immunohistochemistry in lung cancer tissues and adjacent normal lung tissues.Results: We found that Mxi1 is downregulated and correlated with poor prognosis in lung cancer. Using tandem affinity purification technology, we provided evidence that β-Trcp E3 ubiquitin ligase interacts with and promotes the ubiquitination and degradation of Mxi1. Furthermore, we demonstrated that Mxi1 is phosphorylated at S160 site by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase β-Trcp. Moreover, a phosphorylation mutant form of Mxi1 (Mxi1-S160A), which cannot be degraded by S6K1 and β-Trcp, is much more stable and efficient in suppressing the transcriptional activity of Myc and radioresistance in lung cancer cells. More importantly, a strong inverse correlation between S6K1 and Mxi1 expression was observed in human lung cancer tissues.Conclusion: Our findings not only establish a crosstalk between the mTOR/S6K1 signaling pathway and Myc activation, but also suggest that targeting S6K1/Mxi1 pathway is a promising therapeutic strategy for the treatment of lung cancer.

Vaccinia virus protein A49 activates Wnt signalling by targetting the E3 ligase β-TrCP.

Vaccinia virus (VACV) encodes multiple proteins inhibiting the NF-κB signalling pathway. One of these, A49, targets the E3 ubiquitin ligase β-TrCP, which is responsible for the ubiquitylation and consequential proteosomal degradation of IκBα and the release of the NF-κB heterodimer. β-TrCP is a pleiotropic enzyme ubiquitylating multiple cellular substrates, including the transcriptional activator β-catenin. Here we demonstrate that A49 can activate the Wnt signalling pathway, a critical pathway that is involved in cell cycle and cell differentiation, and is controlled by β-catenin. The data presented show that the expression of A49 ectopically or during VACV infection causes accumulation of β-catenin, and that A49 triggering of Wnt signalling is dependent on binding β-TrCP. This is consistent with A49 blocking the ability of β-TrCP to recognise β-catenin and IκBα, and possibly other cellular targets. Thus, A49 targetting of β-TrCP affects multiple cellular pathways, including the NF-κB and Wnt signalling cascades.

Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitin-dependent proteolysis

Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase β-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether β-TrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that β-TrCP downregulated cellular levels of endogenous USP33. We also revealed that β-TrCP interacted with USP33 independently of the classic binding motif for β-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are required for the interaction between β-TrCP and USP33. Consequently, β-TrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase β-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis.

Model of the Interaction between the NF-κB Inhibitory Protein p100 and the E3 Ubiquitin Ligase β-TrCP based on NMR and Docking Experiments.

NF-κB is a major transcription factor whose activation is triggered through two main activation pathways: the canonical pathway involving disruption of IκB-α/NF-κB complexes and the alternative pathway whose activation relies on the inducible proteolysis of the inhibitory protein p100. One central step controlling p100 processing consists in the interaction of the E3 ubiquitin ligase β-TrCP with p100, thereby leading to its ubiquitinylation and subsequent either complete degradation or partial proteolysis by the proteasome. However, the interaction mechanism between p100 and β-TrCP is still poorly defined. In this work, a diphosphorylated 21-mer p100 peptide model containing the phosphodegron motif was used to characterize the interaction with β-TrCP by NMR. In parallel, docking simulations were performed in order to obtain a model of the 21P-p100/β-TrCP complex. Saturation transfer difference (STD) experiments were performed in order to highlight the residues of p100 involved in the interaction with the β-TrCP protein. These results highlighted the importance of pSer865 and pSer869 residues in the interaction with β-TrCP and particularly the Tyr867 that fits inside the hydrophobe β-TrCP cavity with the Arg474 guanidinium group. Four other arginines, Arg285, Arg410, Arg431, and Arg521, were found essential in the stabilization of p100 on the β-TrCP surface. Importantly, the requirement for these five arginine residues of β-TrCP for the interaction with p100 was further confirmed in vivo, thereby validating the docking model through a biological approach.

Regulation of mitosis-meiosis transition by the ubiquitin ligase β-TrCP in male germ cells.

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that β-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of β-TrCP2 in male germ cells of β-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The β-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in β-TrCP-deficient testes. DMRT1 contains a consensus β-TrCP degron sequence that was found to bind β-TrCP. Overexpression of β-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in β-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that β-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation.

Inactivation of ABL kinases suppresses non-small cell lung cancer metastasis.

Current therapies to treat non-small cell lung carcinoma (NSCLC) have proven ineffective owing to transient, variable, and incomplete responses. Here we show that ABL kinases, ABL1 and ABL2, promote metastasis of lung cancer cells harboring EGFR or KRAS mutations. Inactivation of ABL kinases suppresses NSCLC metastasis to brain and bone, and other organs. ABL kinases are required for expression of prometastasis genes. Notably, ABL1 and ABL2 depletion impairs extravasation of lung adenocarcinoma cells into the lung parenchyma. We found that ABL-mediated activation of the TAZ and β-catenin transcriptional coactivators is required for NSCLC metastasis. ABL kinases activate TAZ and β-catenin by decreasing their interaction with the β-TrCP ubiquitin ligase, leading to increased protein stability. High-level expression of ABL1, ABL2, and a subset of ABL-dependent TAZ- and β-catenin-target genes correlates with shortened survival of lung adenocarcinoma patients. Thus, ABL-specific allosteric inhibitors might be effective to treat metastatic lung cancer with an activated ABL pathway signature.

Abstract 1052: Stearoyl-CoA-Desaturase (SCD1) regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ

Abstract One of the most common traits of cancer is the radical change of cellular metabolism. A distinctive aspect of this altered metabolic status is the presence of a larger pool of monounsaturated fatty acids (MUFA), the precursors of the components of cell membranes necessary to sustained the rapidly dividing cancer cells. MUFA are largely generated from saturated fatty acids by the action of the two Stearoyl-CoA desaturases isoforms, namely SCD1 and SCD5. Recent evidences suggest that the major isoform SCD1 plays a role in several cancers. Our group has previously reported, using primary adenocarcinoma cell lines from malignant pleural effusions, that SCD1 inhibition selectively kills ALDH positive cells, a marker of cancer stem cells, causes spheroid 3D cultures collapse in vitro and impairs their growth in vivo. These results suggest that SCD1 may be a critical target in lung cancer tumor-initiating cells and further studies are needed to assess the role of this enzyme in lung cancer. An increasing number of literature points to the Hippo (and their effectors YAP/ TAZ) and to the Wnt/β-catenin signaling pathways as key factors in the development of cancer. Notably, SCD1 has been shown to be involved in the secretion and maturation of active wnt ligands, contributing to the activation of the β-catenin signaling. We report here, using primary cell cultures from adenocarcinoma of the lung, that YAP and TAZ are required for the generation of 3D cultures, as silencing of both genes completely abrogated the capability of generating lung cancer spheroids. Moreover, simultaneously silencing of YAP and TAZ reduced the mRNA levels of OCT4, Nanog and ALDH. We demonstrated that both silencing of SCD1 and its pharmacological inhibition determined a decrease in the expression and nuclear localization of both YAP and TAZ. Moreover, when SCD1 was inhibited, YAP and TAZ transcriptional activity was reduced. We also demonstrated that SCD1 blockade induced a dramatic reduction of the Axin2 mRNA level, one of the β-catenin target genes and a decreased in the β-catenin transcriptional activity, without affecting β-catenin protein expression. YAP and TAZ downregulation induced by SCD1 blockade could be rescued by the addition of exogenous wnt3a ligand, thus indicating that the active β-catenin signaling is necessary for YAP and TAZ stabilization. Ultimately, we observed that the ubiquitin ligase β-TrcP is involved in the SCD1 mediated degradation of YAP and TAZ. These data, overall, demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway and β-catenin pathway in lung cancer 3D spheroid cultures, pointing to lipid metabolism as regulator of cancer stem features. Moreover, these results can be the starting point for further studies focused on the regulation of SCD1 in cancer stem cells, suggesting a new perspective for improving chemotherapeutic responses in cancer treatment, centered on SCD1 inhibition. Citation Format: Alessia Noto, Maria Elena Pisanu, Claudia De Vitis, Giovanni Sorrentino, Giannino Del Sal, Alfredo Budillon, Gennaro Ciliberto, Rita Mancini. Stearoyl-CoA-Desaturase (SCD1) regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1052.

REMOVED: Prenatal stress increased Snk Polo-like kinase 2 and the SCF β-TrCP ubiquitin ligase in the hippocampus of the adult offspring

This article has been removed: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our‐business/policies/article‐withdrawal)This meeting abstract has been removed by the Publisher. Due to an administrative error, abstracts that were not presented at the ISDN 2014 meeting were inadvertently published in the meeting's abstract supplement. The Publisher apologizes to the authors and readers for this error.

HIV Vpu Interferes with NF-κB Activity but Not with Interferon Regulatory Factor 3.

The accessory HIV protein Vpu inhibits a number of cellular pathways that trigger host innate restriction mechanisms. HIV Vpu-mediated degradation of tetherin allows efficient particle release and hampers the activation of the NF-κB pathway thereby limiting the expression of proinflammatory genes. In addition, Vpu reduces cell surface expression of several cellular molecules such as newly synthesized CD4. However, the role of HIV Vpu in regulating the type 1 interferon response to viral infection by degradation of the interferon regulatory factor 3 (IRF3) has been subject of conflicting reports. We therefore systematically investigated the expression of IRF3 in primary CD4(+) T cells and macrophages infected with HIV at different time points. In addition, we also tested the ability of Vpu to interfere with innate immune signaling pathways such as the NF-κB and the IRF3 pathways. We report here that HIV Vpu failed to degrade IRF3 in infected primary cells. Moreover, we observed that HIV NL4.3 Vpu had no effect on IRF3-dependent gene expression in reporter assays. On the other hand, HIV NL4.3 Vpu downmodulated NF-κB-dependent transcription. Mutation of two serines (positions 52 and 56) involved in the binding of NL4.3 Vpu to the βTrCP ubiquitin ligase abolishes its ability to inhibit NF-κB activity. Taken together, these results suggest that HIV Vpu regulates antiviral innate response in primary human cells by acting specifically on the NF-κB pathway. HIV Vpu plays a pivotal role in enhancing HIV infection by counteraction of Tetherin. However, Vpu also regulates host response to HIV infection by hampering the type 1 interferon response. The molecular mechanism by which Vpu inhibits the interferon response is still controversial. Here we report that Vpu affects interferon expression by inhibiting NF-κB activity without affecting IRF3 levels or activity. These data suggest that Vpu facilitates HIV infection by regulating NF-κB transcription to levels sufficient for viral transcription while limiting cellular responses to infection.

Datasets from an interaction proteomics screen for substrates of the SCF(βTrCP) ubiquitin ligase.

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8, 10.1126/scisignal.2005882.

Putative E3 ubiquitin ligase of human rotavirus inhibits NF-κB activation by using molecular mimicry to target β-TrCP.

ABSTRACT NF-κB plays a critical role in the induction and maintenance of innate and adaptive immune transcriptional programs. An associated inhibitor of κB protein (IκB) regulates NF-κB activation and contains a degron motif (DSGΦxS) that undergoes phosphorylation following pathogen recognition or other proinflammatory signals. The E3 ubiquitin ligase SCFβ-TrCP recognizes this phosphodegron through its β-transducin repeat-containing protein (β-TrCP) subunit and induces IκB degradation, allowing NF-κB to translocate to the nucleus and modulate gene expression. Rotavirus (RV), a major cause of pediatric gastroenteritis, can block NF-κB activation through the action of its nonstructural protein NSP1, a putative E3 ubiquitin ligase that mediates the degradation of β-TrCP or other immunomodulatory proteins in a virus strain-specific manner. Here, we show that NSP1 targets β-TrCP by mimicking the IκB phosphodegron. The NSP1 proteins of most human and porcine RV strains conserve a C-terminal phosphodegron-like (PDL) motif, DSGΦS. Deletion of this motif or mutation of its serine residues disrupts NSP1-mediated degradation of β-TrCP and inhibition of NF-κB activation. Additionally, a point mutation within the phosphodegron-binding pocket protects β-TrCP from NSP1-mediated turnover. Fusion of the PDL motif to an NSP1 protein known to target other immunomodulatory proteins generates a chimeric NSP1 protein that can induce β-TrCP degradation and block NF-κB activation. Other viral proteins (Epstein-Barr virus LMP1, HIV-1 Vpu, and vaccinia virus A49) also contain a PDL motif and interact with β-TrCP to inhibit NF-κB activation. Taken together, these data suggest that targeting β-TrCP by molecular mimicry may be a common strategy used by human viruses to evade the host immune response.IMPORTANCE The transcription factor NF-κB, a central regulator of the host response to infection, is a frequent target of viral antagonism. Pathogen detection activates NF-κB by inducing the phosphorylation of an associated inhibitor protein (IκB), which targets IκB for degradation by the E3 ubiquitin ligase β-TrCP. Rotavirus, a significant cause of childhood gastroenteritis, antagonizes NF-κB through the activity of its NSP1 protein, a putative E3 ubiquitin ligase that mediates β-TrCP turnover. Here, we show that NSP1 functions by mimicking the IκB phosphodegron recognized by β-TrCP. Nearly all human rotavirus strains conserve this motif at the NSP1 C terminus, and its removal disrupts NSP1 antagonist activity. This sequence conserves the biochemical properties of the IκB phosphodegron and can rescue antagonist activity when fused to an NSP1 protein otherwise inactive against β-TrCP. Other viral proteins also mimic IκB to disrupt NF-κB activation, indicating that this is an important immune evasion strategy.

Phosphorylation-dependent ubiquitination of paraxial protocadherin (PAPC) controls gastrulation cell movements.

Paraxial protocadherin (PAPC) has been shown to be involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation. PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP. We also show that precise control of PAPC by phosphorylation/ubiquitination is essential for normal Xenopus gastrulation cell movements. Taken together, our findings unveil a novel mechanism of regulation of a cell adhesion protein and show that this system plays a crucial role in vertebrate embryogenesis.

The Oncogenic STP Axis Promotes Triple-Negative Breast Cancer via Degradation of the REST Tumor Suppressor

Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 ("STP axis") cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC.

PLK1 and β-TrCP-dependent ubiquitination and degradation of Rap1GAP controls cell proliferation.

Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.

Oncogenic Y641 mutations in EZH2 prevent Jak2/β-TrCP-mediated degradation

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase β-TrCP (FBXW1). β-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs β-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of β-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind β-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that β-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

SCF-β-TrCP E3 Ubiquitin Ligase-Mediated Proteolysis Of EZH2 Regulates H3K27 Trimethylation Activity In Germinal Center-Derived B-Cell Lymphomas

EZH2 is a critical enzymatic subunit of the PRC2 which trimethylates histone H3 (H3K27) to mediate gene repression. While overexpression of EZH2 and its increased H3K27 methylation have generally been associated with hematologic malignancies and solid tumors, recurrent gain of function somatic mutations in EZH2 have been recently reported in germinal center B-cell (GCB) derived lymphomas. These mutations, which replace a single tyrosine in the SET domain of the EZH2 protein (Y641), occur in 7.2% of FLs (follicular lymphoma) and 21.7% of GCB DLBCL (diffuse large B-cell lymphoma) and are absent from ABC DLBCLs. Consequently, B-cell lymphoma cell lines and lymphoma primary samples harboring heterozygous EZH2Y641mutations exhibit increased levels of histone H3 Lys27 trimethylation (H3K27me3) and promote a lymphoproliferative phenotype with expansion of the GC B cell compartment.Although extensive studies have been conducted to understand the role of polycomb group of proteins in cancer pathogenesis, few studies have been aimed at understanding the post-translational regulation of these proteins. Skp1/Cullin1/F- box protein (SCF) ubiquitin ligase complexes are the largest family of multicomponent ring E3 ubiquitin ligases that control the degradation of many important regulatory proteins and have been implicated in the regulation of epigenetic regulators. However, the mechanism of EZH2 degradation and its implication in PRC2 mediated methyl transferase activity in a cancer-specific context is not fully understood.Cullin-ring ubiquitin ligases (CRLs) form the largest known class of multicomponent E3 ubiqutin ligase family in eukaryotes and target a wide array of substrates involved in several biological processes. To investigate a CRL-mediated basis for post-translational EZH2 regulation, we screened a panel of cullin family E3 ligases for interaction with EZH2 and discovered that EZH2 was immunoprecipitated by cullin1 exclusively among all other cullins (cul2,3,4a,4b,5 and 7). We engineered and expressed a cullin1 dominant negative protein which resulted in EZH2 accumulation in a dose dependent manner. Using co-immunoprecipitation approach, we screened 8 different F box proteins and observed that SCF E3 ubiquitin ligase β-TrCP (FBXW1) uniquely and specifically interacted with EZH2. Further, RNAi-mediated silencing of β-TrCP resulted in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the Y641 mutants recurrently identified and relevant in lymphoma pathogenesis were unable to bind β-TrCP. Further, cycloheximide chase experiments showed that Y641 mutants endogenously expressed in lymphoma-derived cells exhibit increased EZH2 stability as well as enhanced H3K27 trimethylation activity.Our studies demonstrate that β-TrCP plays an important role in controlling H3K27 trimethylation activity via an unusual recognition mechanism targeting EZH2 for proteasomal degradation. Our findings delineate a novel β-TrCP/EZH2 axis requiring an intact Y641 residue for EZH2 regulation by β-TrCP. Oncogenic mutations in Y641 in GCB-cell derived lymphomas confer increased stability to EZH2 and render it resistant to β-TrCP-mediated degradation. This newly identified mechanism might help in design potential novel therapeutic approaches for clinical management of cancer potentially driven by aberrant accumulation/function of EZH2. Disclosures:No relevant conflicts of interest to declare.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Prenatal stress increased Snk Polo-like kinase 2, SCF β-TrCP ubiquitin ligase and ubiquitination of SPAR in the hippocampus of the offspring at adulthood

Exposure to excessive glucocorticoids during fetal development period contributes to later life psychopathology. Prenatal stress decreases dendritic spine density and impair LTP in the hippocampus of rat pups, however, the mechanisms regulating these changes are still unclear.Glutamate receptors are localized in the postsynaptic density. PSD-95 is a postsynaptic scaffolding protein that plays a role in synaptic maturation and regulation of the synaptic strength and plasticity. PSD-95 interacts with other proteins to form the protein networks that promote dendritic spine formation. The present study investigated the effect of prenatal stress on the levels of scaffolding proteins of NMDA receptor in the hippocampus in order to explain how prenatal stress alters the amount of NMDA receptor in the pups’ brain. Pregnant rats were randomly assigned to either the prenatal stress (PS) or the control group (C). The pregnant rats in the PS group were restrained in a plexiglas restrainer for 4h/day during the GD 14–21. Control rats were left undisturbed for the duration of their pregnancies. The amount of PSD-95, SPAR, NR2A and NR2B, as well as the levels of Snk Polo-like kinase 2 and the SCF β-TrCP ubiquitin ligase were measured in the hippocampus of the offspring. The results show that prenatal stress induces a reduction in the amount of NR2B and NR2A subunits in the hippocampus of rat pups, parallel to the decrease in PSD-95 and SPAR at P40 and P60. Moreover, prenatal stress increases Snk and β-TrCP in the hippocampus of rat pups, and the timing correlates with the decrease of SPAR and PSD-95. Prenatal stress also induces a significantly increases in the level of ubiquitinated SPAR in the hippocampus of rat pups at adulthood. The results suggest that degradation of SPAR via UPS system may contribute to the loss of PSD-95 and NMDA receptor subunits in the hippocampus of rat pups at adulthood. In conclusion, the present work demonstrates that the developing brain is critically influenced by glucocorticoids, especially during pre- and early postnatal period, which can have long-term effects on brain development. In addition, an involvement of the UPS system in the prenatal stress model has led to a greater understanding of the effects of prenatal stress later on in life.

TRIB2 Acts Downstream of Wnt/TCF in Liver Cancer Cells to Regulate YAP and C/EBPα Function

Dysregulation of Wnt signaling is closely associated with human liver tumorigenesis. However, liver cancer-specific Wnt transcriptional programs and downstream effectors remain poorly understood. Here, we identify tribbles homolog 2 (TRIB2) as a direct target of Wnt/TCF in liver cancer and demonstrate that transcription of Wnt target genes, including TRIB2, is coordinated by the TCF and FoxA transcription factors in liver cancer cells. We show that Wnt-TRIB2 activation is critical for cancer cell survival and transformation. Mechanistically, TRIB2 promotes protein stabilization of the YAP transcription coactivator through interaction with the βTrCP ubiquitin ligase. Furthermore, we find that TRIB2 relieves the liver tumor suppressor protein C/EBPα-mediated inhibition of YAP/TEAD transcriptional activation in liver cancer cells. Altogether, our study uncovers a regulatory mechanism underlying liver cancer-specific Wnt transcriptional output, and suggests that TRIB2 functions as a signaling nexus to integrate the Wnt/β-catenin, Hippo/YAP, and C/EBPα pathways in cancer cells.