CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation

The human inhibitor of Bruton''s tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3^IBTK complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3^IBTK for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3^IBTK regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5′-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3^IBTK as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.     

Regulation of RNF144A E3 Ubiquitin Ligase Activity by Self-association through Its Transmembrane Domain

RNF144A, an E3 ubiquitin ligase for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), can promote DNA damage-induced cell apoptosis. Here we characterize an important regulation of RNF144A through its transmembrane (TM) domain. The TM domain of RNF144A is highly conserved among species. Deletion of the TM domain abolishes its membrane localization and also significantly reduces its ubiquitin ligase activity. Further evidence shows that the TM domain is required for RNF144A self-association and that the self-association may be partially mediated through a classic GXXXG interaction motif. A mutant RNF144A-G252L/G256L (in the G²⁵²XXXG²⁵⁶ motif) preserves membrane localization but is defective in self-association and ubiquitin ligase activity. On the other hand, a membrane localization loss mutant of RNF144A still retains self-association and E3 ligase activity, which can be blocked by additional G252L/G256L mutations. Therefore, our data demonstrate that the TM domain of RNF144A has at least two independent roles, membrane localization and E3 ligase activation, to regulate its physiological function. This regulatory mechanism may be applicable to other RBR (RING1-IBR-RING2) E3 ubiquitin ligases because, first, RNF144B also self-associates. Second, all five TM-containing RBR E3 ligases, including RNF144A and RNF144B, RNF19A/Dorfin, RNF19B, and RNF217, have the RBR-TM(GXXXG) superstructure. Mutations of the GXXXG motifs in RNF144A and RNF217 have also be found in human cancers, including a G252D mutation of RNF144A. Interestingly, RNF144A-G252D still preserves self-association and ubiquitin ligase activity but loses membrane localization and is turned over rapidly. In conclusion, both proper membrane localization and self-association are important for RNF144A function.     

Hypoxia-inducible CircPFKFB4 Promotes Breast Cancer Progression by Facilitating the CRL4DDB2 E3 Ubiquitin Ligase-mediated p27 Degradation

Hypoxic microenvironment and circular RNAs (circRNAs) have shown critical implications in breast cancer (BC) progression. However, the specific functions and underlying mechanisms of circRNAs in BC under hypoxia remain largely unknown. We first screened for differentially expressed circRNAs in normoxic and hypoxic MCF-7 cells using circRNA microarray. A novel hypoxia-induced circRNA, circPFKFB4, was identified. Clinical investigation showed that circPFKFB4 was highly expressed in BC tissues and cell lines, and its overexpression was positively correlated with the advanced clinical stage and poor prognosis of BC patients. Functionally, circPFKFB4 promoted the proliferation of BC cells both in vitro and in vivo. Mechanistically, HIF1α bound to hypoxia response elements in the promoter region of the PFKFB4 gene to facilitate the biogenesis of circPFKFB4 under hypoxia. Hypoxia-induced circPFKFB4 directly bound to both DDB1 and DDB2 and promoted the CRL4^DDB2 E3 ubiquitin ligase assembly, resulting in p27 ubiquitination and BC progression under hypoxia. Our findings revealed a novel interaction between circPFKFB4 and the CRL4^DDB2 E3 ubiquitin ligase, suggesting that circPFKFB4 might serve as a promising biomarker and therapeutic target for BC.     

The Wnt Signaling Antagonist Dapper1 Accelerates Dishevelled2 Degradation via Promoting Its Ubiquitination and Aggregate-induced Autophagy

Autophagy is a regulated process that sequesters and transports cytoplasmic materials such as protein aggregates via autophagosomes to lysosomes for degradation. Dapper1 (Dpr1), an interacting protein of Dishevelled (Dvl), antagonizes Wnt signaling by promoting Dishevelled degradation via lysosomes. However, the mechanism is unclear. Here, we show that Dpr1 promotes the von Hippel-Lindau tumor suppressor (VHL)-mediated ubiquitination of Dvl2 and its autophagic degradation. Knockdown of Dpr1 decreases the interaction between Dvl2 and pVHL, resulting in reduced ubiquitination of Dvl2. Dpr1-mediated autophagic degradation of Dvl2 depends on Dvl2 aggregation. Moreover, the aggregate-prone proteins Dvl2, p62, and the huntingtin mutant Htt103Q promote autophagy in a Dpr1-dependent manner. These protein aggregates enhance the Beclin1-Vps34 interaction and Atg14L puncta formation, indicating that aggregated proteins stimulate autophagy initiation. Ubiquitination is not essential for the aggregate-induced autophagy initiation as inhibition of the ubiquitin-activation E1 enzyme activity did not block the aggregate-induced Atg14L puncta formation. Our findings suggest that Dpr1 promotes the ubiquitination of Dvl2 by pVHL and mediates the protein aggregate-elicited autophagy initiation.     

Post-translational control of the long and winding road to cholesterol

The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin–proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.     

HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest

Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G₂/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G₂/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G₂/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G₂/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.     

NRBP1-Containing CRL2/CRL4A Regulates Amyloid β Production by Targeting BRI2 and BRI3 for Degradation

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IKBKE protein activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and the pleckstrin homology domain to sustain malignant transformation

Serine/threonine kinase Akt regulates key cellular processes such as cell growth, proliferation, and survival. Activation of Akt by mitogenic factor depends on phosphatidylinositol 3-kinase (PI3K). Here, we report that IKBKE (also known as IKKε and IKKi) activates Akt through a PI3K-independent pathway. IKBKE directly phosphorylates Akt-Thr308 and Ser473 independent of the pleckstrin homology (PH) domain. IKBKE activation of Akt was not affected by inhibition of PI3K, knockdown of PDK1 or mTORC2 complex. Further, this activation could be inhibited by Akt inhibitors MK-2206 and GSK690693 but not the compounds (perifosine and triciribine) targeting the PH domain of Akt. Expression of IKBKE largely correlates with activation of Akt in breast cancer. Moreover, inhibition of Akt suppresses IKBKE oncogenic transformation. These findings indicate that IKBKE is an Akt-Thr308 and -Ser473 kinase and directly activates Akt independent of PI3K, PDK1, and mTORC2 as well as PH domain. Our data also suggest that Akt inhibitors targeting the PH domain have no effect on the tumors in which hyperactive Akt resulted from elevated IKBKE.     

Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration

Healthy mitochondria are required in cell metabolism and deregulation of underlying mechanisms is often involved in human diseases and neurological disorders. Post-translational modifications of mitochondrial proteins regulate their function and activity, accordingly, impairment of ubiquitin proteasome system affects mitochondria homeostasis and organelle dynamics. In the present study we have investigated the role of the ubiquitin protease Ubp8 in S. cerevisiae respiration. We show that Ubp8 is necessary for respiration and its expression is upregulated in glycerol respiratory medium. In addition, we show that the respiratory defects in absence of Ubp8 are efficiently rescued by disruption of the E3 Ub-ligase Psh1, suggesting their epistatic link. Interestingly, we found also that Ubp8 is localized into mitochondria as single protein independently of SAGA complex assembly, thus suggesting an independent function from the nuclear one. We also show evidences on the importance of HAT Gcn5 in sustaining Ubp8 expression and affecting the amount of protein in mitochondria. Collectively, our results have investigated the role of Ubp8 in respiratory metabolism and highlight the role of ubiquitin related pathways in the mitochondrial functions of S. cerevisiae.     

沉默脂肪细胞增强子结合蛋白2通过PI3K/Akt途径抑制肝癌细胞增殖和迁移

脂肪细胞增强子结合蛋白2(AEBP2)作为多梳抑制复合物2(PRC2)的组成蛋白质,参与多种肿瘤细胞的增殖和迁移,然而其在肝癌中的作用尚不清楚。本研究基于UALCAN和Kaplan-Meier Plotter数据库分析发现,AEBP2在肝癌组织中高表达,并且与患者的不良预后呈正相关。实时荧光定量PCR和蛋白质印迹结果证实,AEBP2在肝癌细胞中的表达高于正常肝细胞。在HepG2和Huh-7细胞中转染AEBP2 siRNA,平板克隆、CCK-8、流式细胞术、划痕愈合和Transwell结果显示,沉默AEBP2可以抑制肝癌细胞增殖、迁移和侵袭,并促进细胞凋亡(P＜0.05)。免疫荧光检测和蛋白质印迹结果显示,沉默AEBP2能够抑制肝癌细胞上皮-间质转化(EMT)(P＜0.05)。生物信息学分析结果表明,AEBP2参与调控PI3K/Akt信号通路。蛋白质印迹结果证实,沉默AEBP2能下调PI3K、p-AKT (S473)、mTOR、MMP-2和MMP-9的蛋白质表达水平(P＜0.05)。此外,沉默AEBP2对HepG2细胞迁移和侵袭的影响可被PI3K/Akt通路激动剂胰岛素样生长因子1(IGF-1)部分逆转(P＜0.01)。综上所述,AEBP2可能通过调节PI3K/Akt途径促进肝癌细胞增殖和迁移。本研究为AEBP2在肝癌中的作用提供理论依据。     

Wnt3a regulates proliferation,apoptosis and function of pancreatic NIT‐1 beta cells via activation of IRS2/PI3K signaling

Wnt‐signaling pathway is implicated in pancreatic development and functional regulation of mature beta‐cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta‐cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta‐cell line (NIT‐1), we examined the role of IRS2/PI3K in the mediation of Wnt3a‐stimulated beta‐cell growth. Real‐time PCR and Western blot were employed to investigate the activity of Wnt/β‐catenin and IRS2/PI3K signaling. Proliferation of NIT‐1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/β‐catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT‐1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT‐1 cell proliferation, inhibited cytokine‐induced beta‐cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT‐1 beta cells via activation of Wnt/β‐catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta‐cells also being IRS2/PI3K/AKT dependent. J. Cell. Biochem. 114: 1488–1497, 2013. © 2013 Wiley Periodicals, Inc.     

The serine/threonine protein kinase of Streptococcus suis serotype 2 affects the ability of the pathogen to penetrate the blood–brain barrier

Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes meningitis in humans and pigs. However, the mechanism whereby SS2 crosses the microvasculature endothelium of the brain is not understood. In this study, transposon (TnYLB‐1) mutagenesis was used to identify virulence factors potentially associated with invasive ability in pathogenic SS2. A poorly invasive mutant was identified and was found to contain a TnYLB‐1 insertion in the serine/threonine kinase (stk) gene. Transwell chambers containing hBMECs were used to model the blood–brain barrier (BBB). We observed that the SS2 wild‐type ZY05719 strain crossed the BBB model more readily than the mutant strain. Hence, we speculated that STK is associated with the ability of crossing blood–brain barrier in SS2. In vitro, compared with ZY05719, the ability of the stk‐deficient strain (Δstk) to adhere to and invade both hBMECs and bEnd.3 cells, as well as to cross the BBB, was significantly attenuated. Immunocytochemistry using antibodies against claudin‐5 in bEnd.3 cells showed that infection by ZY05719 disrupted BBB tight junction proteins to a greater extent than in infection by Δstk. The studies revealed that SS2 initially binds at or near intercellular junctions and crosses the BBB via paracellular traversal. Claudin‐5 mRNA levels were indistinguishable in ZY05719‐ and Δstk‐infected cells. This result indicated that the decrease of claudin‐5 was maybe induced by protein degradation. Cells infected by ZY05719 exhibited higher ubiquitination levels than cells infected by Δstk. This result indicated that ubiquitination was involved in the degradation of claudin‐5. Differential proteomic analysis showed that E3 ubiquitin protein ligase HECTD1 decreased by 1.5‐fold in Δstk‐infected bEnd.3 cells relative to ZY05719‐infected cells. Together, the results suggested that STK may affect the expression of E3 ubiquitin ligase HECTD1 and subsequently increase the degradation of claudin‐5, thus enabling SS2 to traverse the BBB.     

前列腺素E2及其受体在链脲佐菌素诱导的糖尿病大鼠视网膜病变中的作用及机制研究

摘要 目的：探究前列腺素E2(prostaglandin E2,PGE2)及其受体前列腺素E2受体（E-prostanoid2 receptor,EP2R）在链脲佐菌素诱导的糖尿病大鼠视网膜病变中的作用及机制。方法：将SD大鼠随机分为6组：对照组使用标准饲料喂养;其他组大鼠使用高脂高糖饲料喂养+腹膜内注射链脲佐菌素(30 mg/kg)建立糖尿病大鼠模型;PGE2组、Butaprost组、AH6809组大鼠分别给予玻璃体腔内注射5 mmol/L的PGE2、EP2R激动剂Butaprost或EP2R抑制剂AH6809,注射剂量为6 μL。DMSO组注射等剂量DMSO盐溶液。每周注射1次,共注射4周。通过苏木精伊红(HE)染色评价视网膜病变;免疫组化或蛋白质印迹分析视网膜组织中EP2R、胰岛素受体底物1 (IRS-1)、磷酸肌醇3激酶(PI3K)、p-PI3K、蛋白激酶B (Akt)、p-Akt、细胞间粘附分子-1 (ICAM-1)、内皮一氧化氮合酶(eNOS)、核因子κB p65 (NF-κB p65)和血管内皮生长因子(VEGF)的表达。此外,分别应用PGE2、Butaprost或AH6809处理高糖培养基(4.5 g/L葡萄糖)培养的视网膜微血管内皮细胞系(HRMEC),并检测各组细胞活力、细胞凋亡率和血管生成情况。结果：与正常视网膜组织相比,糖尿病大鼠视网膜组织中EP2R呈显著高表达(P<0.05)。与对照组和模型组相比,PGE2和Butaprost组的EP2R、IRS-1、p-PI3K、p-Akt、ICAM-1、eNOS、NF-κBp65和VEGF的表达水平显著升高,而AH6809组的上述蛋白的表达水平显著降低(P<0.05)。体外研究中,与对照组和模型组HRMEC相比,PGE2和Butaprost处理的HRMEC的活力和血管生成数量显著升高,而细胞凋亡率显著降低,AH6809处理则抑制了上述细胞改变(P<0.05)。结论：PGE2/EP2R可能通过促进IRS-1/PI3K/Akt信号通路介导的炎症反应、细胞凋亡和血管生成促进糖尿病视网膜病变的发生和发展。     

Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer

Although E3 ubiquitin ligases are deemed to play key roles in normal cell function and homeostasis, whether their alterations contribute to cancer pathogenesis remains unclear. In this study, we sought to investigate potential mechanisms that govern WWP1/Tiul1 (WWP1) ubiquitin ligase activity, focusing on its ability to trigger degradation of TGFβ type I receptor (TβRI) in conjunction with Smad7. Our data reveal that the WWP1 protein is very stable at steady states because its autopolyubiquitination activity is silenced due to an intra-interaction between the C2 and/or WW and Hect domains that favors WWP1 monoubiquitination at the expense of its polyubiquitination or polyubiquitination of TβRI. Upon binding of WWP1 to Smad7, this functional interplay is disabled, switching its monoubiquitination activity toward a polyubiquitination activity, thereby driving its own degradation and that of TβRI as well. Intriguingly, a WWP1 point mutation found in human prostate cancer disrupts this regulatory mechanism by relieving the inhibitory effects of C2 and WW on Hect and thereby causing WWP1 hyperactivation. That cancer-driven alteration of WWP1 culminates in excessive TβRI degradation and attenuated TGFβ cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1.     

Cdh1, a Substrate-recruiting Component of Anaphase-promoting Complex/Cyclosome (APC/C) Ubiquitin E3 Ligase,Specifically Interacts with Phosphatase and Tensin Homolog (PTEN) and Promotes Its Removal from Chromatin

A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on Ser-380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains, and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on Ser-380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, the PTEN interaction with Cdh1 was phosphatase-independent, and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.     

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 (^S730AVACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with ^S730AVACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with ^S730AVACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.