FBXW7 is involved in Aurora B degradation

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.     

Differential regulation of FBXW7 isoforms by various stress stimuli

Fbxw7 is a tumor suppressor mutated in a wide range of human cancers. It serves as the substrate recognition component of SCF E3 ubiquitin ligases, and intensive effort was made to identify its substrates. Some of the substrates are central regulators of the cell cycle, cell fate determination, and cellular survival. Unlike the many efforts aimed at identifying novel targets, little is known about the regulation of Fbw7 isoform expression. In this study, we examined the mRNA expression of different FBXW7 isoforms during the cell cycle and after exposure to various stress stimuli. We observed that Fbw7β is induced by all the stress stimuli tested, mostly, but not exclusively, in a p53-dependent manner. In fact, FBXW7β was found to be the most potently induced p53 target gene in HCT-116 cells. Expression of FBXWα and γ is p53-independent and their responsiveness to most stress stimuli is limited. Furthermore, their pattern of stress responsiveness is very different from that of the β isoform. Under certain conditions, the same genotoxic agent stimulates induction of β and repression of α. Analysis of FACS-sorted cells in specific phases of the cell cycle by using fluorescent ubiquitination-based cell cycle indicator (FUCCI), showed a significant repression of the γ isoform during the S phase of normal cycling HCT-116 cells. Altogether, this study suggests differential regulation of the 3 Fbw7 isoforms.     

FBXW7 Confers Radiation Survival by Targeting p53 for Degradation

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Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint

The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.     

miR-182靶向抑制FBXW7表达影响非小细胞肺癌细胞增殖研究

目的:研究microRNA-182(miR-182)在非小细胞肺癌(NSCLC)组织中的表达,并探讨其对NSCLC细胞增殖的影响及作用机制。方法:采用实时荧光定量PCR (qRT-PCR)检测miR-182在11例NSCLC及相应癌旁组织中的表达情况;Western blot检测FBXW7,c-Jun,c-Myc及cyclin D蛋白的表达;将miR-182模拟物,抑制物及相应空白对照瞬时转染H460细胞后,以细胞增殖与活性检测和克隆形成实验检测细胞系的增殖情况;流式细胞术检测细胞周期和凋亡变化;荧光素酶报告基因实验证实miR-182对FBXW7的靶向性作用。结果:NSCLC组织中miR-182的相对表达水平显著高于癌旁组织(P0.05)。转染组与对照组相比,H460细胞生长、克隆形成能力显著增强,细胞周期进程加快,细胞凋亡受到抑制(P0.05)。在NSCLC组织中,FBXW7蛋白的表达水平明显低于癌旁组织(P0.05)。miR-182 mimics显著降低野生型FBXW7质粒荧光素酶的活性,然而将结合位点突变后,miR-182 mimics则不再影响荧光素酶的活性。结论:miR-182在NSCLC组织中高表达,与FBXW7之间存在靶向关系,通过下调FBXW7蛋白表达促进NSCLC细胞的增殖,参与肿瘤的发生发展,预示其可能成为一种潜在的生物标志和治疗靶点。     

miR-367 stimulates Wnt cascade activation through degrading FBXW7 in NSCLC stem cells

Lung carcinoma tops the categories of cancer related motility, and has been treated as the main threat to human health. The functions and related mechanism of FBXW7 controlled lung cancer stem cells' signatures is barely unknown, and the miR-367 regulations of FBXW7 via Wnt signaling have not been explored. Cancer stem cells of either ALDH1+ or CD133+ phenotype were found to be referred to advanced stages in patients with NSCLC (non-small cell lung carcinoma). To study the roles of miR-367, we found greater miR-367 level or FBXW7 level was reserved in NSCLC than that of paired adjacent normal tissues, and their upregulations were positively correlated with Wnt signaling activation. On the contrary, increased miR-367 was correlated with Let-7 repression. MiR-367 was related to stronger sphere forming ability in stem cells of NSCLC. We then explored the functions of the endogenous miR-367 in stem-like cells isolated from NSCLC cell lines. In HEK-293 cells, we identified FBXW7 as the direct downstream gene of miR-367, which consequently released the LIN-28 dependent inhibition of suppressive Let-7. Through informatics analysis, miR-367 was predicated to function through Wnt signaling, and decreased Let-7 played the pivotal role to maintain TCF-4/Wnt pathway activity. The reintroduction of FBXW7 abolished the oncogenic stimulation of miR-367 on TCF-4 activity, with Wnt signaling factors depression. In conclusion, our findings demonstrated the oncogenic roles of miR-367 exerting on the self-renewal ability of cancer stem-like cells through degrading the suppressive FBXW7, eventually helping to maintain Wnt signaling activation through a LIN28B/Let-7 dependent manner.     

PML Suppresses Influenza Virus Replication by Promoting FBXW7 Expression

Virologica Sinica - Influenza A viruses (IAV) are responsible for seasonal flu epidemics, which can lead to high morbidity and mortality each year. Like other viruses, influenza virus can hijack...     

塞内卡病毒通过激活自噬降解FBXW7拮抗宿主的抗病毒应答

【背景】塞内卡病毒(seneca valley virus, SVV)可引起猪严重的水疱性疾病,但目前仍无商业化疫苗,解析其与宿主抗病毒分子的相互作用具有重要意义。【目的】探究E3泛素连接酶(F-box and WD repeat domain containing 7, FBXW7)在抗SVV感染中的作用及其机制。【方法】利用小干扰技术敲低PK-15细胞FBXW7,结合RT-qPCR、组织半数感染量(tissue culture infective dose 50%,TCID50)和Western blotting分析FBXW7对SVV在PK-15细胞中复制及细胞因子的影响。【结果】Western blotting结果显示,SVV感染能够抑制PK-15细胞中FBXW7蛋白表达水平;敲低FBXW7后促进SVV复制,同时炎性细胞因子IL-6、IFN-β、TNF-αmRNA水平均显著降低;进一步研究显示,敲低ATG5抑制自噬后抑制FBXW7蛋白降解,SVV复制水平下降。【结论】本研究发现FBXW7对SVV复制具有抑制作用,SVV通过激活自噬介导FBXW7降解,为研发抗病毒靶点提供了新策略。     

有丝分裂激酶Aurora B的研究进展

真核生物细胞通过有丝分裂将遗传物质均等地分配到两个子细胞中,从而维持基因组的稳定性。有丝分裂的每一环节都需要精准而细致的调控,这依赖于一系列调节机制,尤其需要多个相关激酶的共同协调。Aurora B是一个关键的有丝分裂调控激酶,伴随有丝分裂的进行,其先后在染色体臂、内着丝粒、中央纺锤体、中体上动态分布。与其高度时空动态性相一致的是,Aurora B在有丝分裂的多个环节,如姐妹染色体粘连、动粒微管连接、纺锤体检验点和胞质分裂过程中都发挥着一系列重要功能。本文将概述近年来Aurora B激酶功能与调控方面的研究进展。     

The anti-angiogenesis role of FBXW7 in diabetic retinopathy by facilitating the ubiquitination degradation of c-Myc to orchestrate the HDAC2

Diabetic retinopathy (DR) is the most prevalently occurring microvascular complication in diabetic patients that triggers severe visual impairments. The anti-angiogenesis role of FBXW7 has been identified in breast cancer. Therefore, this study intends to decipher the mechanism of FBXW7 in angiogenesis of DR. DR model was induced on mice using high-glucose (HG) and high-fat diet, and retinal microvascular endothelial cells (RMECs) isolated from normal mice were induced with HG, followed by evaluation of FBXW7, Ki67, HIF-1α and VEGF expression by immunofluorescence, immunohistochemistry or Western blot analysis. After gain- and loss-of-function assays in normal and DR mice, angiogenesis was assessed by CD31 fluorescence staining and Western blot analysis. After ectopic expression and silencing experiments in HG-induced RMECs, RMEC proliferation, migration and angiogenesis were, respectively, determined by EdU, Transwell and in vitro angiogenesis assays. The impact of FBXW7 on the ubiquitination of c-Myc was studied by cycloheximide chase assay and proteasome inhibition, and the binding of c-Myc to HDAC2 promoter by dual-luciferase reporter gene experiment. DR mice and HG-induced RMECs possessed down-regulated FBXW7 and up-regulated Ki67, HIF-1α and VEGF. Silencing FBXW7 enhanced angiogenesis in normal mouse retinal tissue, but overexpressing FBXW7 or silencing c-Myc diminished angiogenesis in DR mouse retinal tissue. Overexpressing FBXW7 or silencing c-Myc depressed proliferation, migration and angiogenesis in HG-induced RMECs. FBXW7 induced c-Myc ubiquitination degradation, and c-Myc augmented HDAC2 expression by binding to HDAC2 promoter. Conclusively, our data provided a novel sight of anti-angiogenesis role of FBXW7 in DR by modulating the c-Myc/HDAC2 axis.     

Regulation of the Mdm2–p53 pathway by the ubiquitin E3 ligase MARCH7

The tumor suppressor p53 plays a prominent role in the protection against cancer. The activity of p53 is mainly controlled by the ubiquitin E3 ligase Mdm2, which targets p53 for proteasomal degradation. However, the regulation of Mdm2 remains not well understood. Here, we show that MARCH7, a RING domain‐containing ubiquitin E3 ligase, physically interacts with Mdm2 and is essential for maintaining the stability of Mdm2. MARCH7 catalyzes Lys⁶³‐linked polyubiquitination of Mdm2, which impedes Mdm2 autoubiquitination and degradation, thereby leading to the stabilization of Mdm2. MARCH7 also promotes Mdm2‐dependent polyubiquitination and degradation of p53. Furthermore, MARCH7 is able to regulate cell proliferation, DNA damage‐induced apoptosis, and tumorigenesis via a p53‐dependent mechanism. These findings uncover a novel mechanism for the regulation of Mdm2 and reveal MARCH7 as an important regulator of the Mdm2–p53 pathway.     

The methylation status of FBXW7 beta-form correlates with histological subtype in human thymoma

FBXW7 is reported to be a tumor suppressor gene, and the functional inactivation of FBXW7 has been reported in various human tumors. In this study, we investigated the FBXW7 gene in human thymoma; although no mutations were evident, a significantly high frequency of methylation in the FBXW7 β-form promoter was observed in types B1 or higher (P = 0.014). We propose a novel mechanism for the pathogenesis of thymoma by FBXW7 β-form and hypothesize that expressional suppression plays an important role in the malignant potential of thymoma.     

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent,FBXW7-mediated Ubiquitination and Proteasomal Degradation

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.     

Mps1 promotes rapid centromere accumulation of Aurora B

Aurora B localization to mitotic centromeres, which is required for proper chromosome alignment during mitosis, relies on Haspin-dependent histone H3 phosphorylation and on Bub1-dependent histone H2A phosphorylation-which interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby promoting Aurora B centromere recruitment in early mitosis. Importantly, chromosome biorientation defects caused by Mps1 inhibition are improved by restoring Aurora B centromere recruitment. As Mps1 kinetochore localization reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid centromere accumulation of Aurora B at the onset of mitosis.