Regulation of PTEN degradation and NEDD4–1 E3 ligase activity by Numb

The critical tumor suppressor PTEN is regulated by numerous post-translational modifications including phosphorylation, acetylation and ubiquitination. Ubiquitination of PTEN was reported to control both PTEN stability and nuclear localization. Notably, the HECT E3-ligase NEDD4–1 was identified as the ubiquitin ligase for PTEN, mediating its degradation and down-stream events. However, the mechanisms how NEDD4–1 is regulated by up-stream signaling pathways or interaction with other proteins in promoting PTEN degradation remain largely unclear. In the present study, we identified that the adaptor protein Numb, which is demonstrated to be a novel binding partner of NEDD4–1, plays important roles in controlling PTEN ubiquitination through regulating NEDD4–1 activity and the association between PTEN and NEDD4–1. Furthermore, we provided data to show that Numb regulates cell proliferation and glucose metabolism in a PTEN-dependent manner. Overall, our study revealed a novel regulation of the well-documented NEDD4–1/PTEN pathway and its oncogenic behavior.     

NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN

The tumor suppressor PTEN, a critical regulator for multiple cellular processes, is mutated or deleted frequently in various human cancers. Subtle reductions in PTEN expression levels have profound impacts on carcinogenesis. Here we show that PTEN level is regulated by ubiquitin-mediated proteasomal degradation, and purified its ubiquitin ligase as HECT-domain protein NEDD4-1. In cells NEDD4-1 negatively regulates PTEN stability by catalyzing PTEN polyubiquitination. Consistent with the tumor-suppressive role of PTEN, overexpression of NEDD4-1 potentiated cellular transformation. Strikingly, in a mouse cancer model and multiple human cancer samples where the genetic background of PTEN was normal but its protein levels were low, NEDD4-1 was highly expressed, suggesting that aberrant upregulation of NEDD4-1 can posttranslationally suppress PTEN in cancers. Elimination of NEDD4-1 expression inhibited xenotransplanted tumor growth in a PTEN-dependent manner. Therefore, NEDD4-1 is a potential proto-oncogene that negatively regulates PTEN via ubiquitination, a paradigm analogous to that of Mdm2 and p53.     

Crucial role of the C-terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation

PTEN (phosphatase and tensin homologue deleted on chromosome 10), a potent tumour suppressor and multifunctional signalling protein, is under intricate regulation. In the present study, we have investigated the mechanism and regulation of PTEN ubiquitination catalysed by NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1), a ubiquitin ligase for PTEN we identified recently. Using the reconstituted assay and cellular analysis, we demonstrated that NEDD4-1-mediated PTEN ubiquitination depends on its intact HECT (homologous to E6-associated protein C-terminus) domain. Instead of using its WW domains (protein-protein interaction domains containing two conserved tryptophan residues) as a protein interaction module, NEDD4-1 interacts with PTEN through its N-terminal region containing a C2 domain as well as the HECT domain. Strikingly, we found that a C-terminal truncated PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN, suggesting an intrinsic inhibitory effect of the PTEN C-terminus on PTEN-NEDD4-1 interaction. Moreover, the C-terminal truncated PTEN is more sensitive to NEDD4-1-mediated ubiquitination and degradation. Therefore the present study reveals that the C-terminus of PTEN plays a critical role in stabilizing PTEN via antagonizing NEDD4-1-induced PTEN protein decay; conversely, truncation of the PTEN C-terminus results in rapid NEDD4-1-mediated PTEN degradation, a possible mechanism accounting for attenuation of PTEN function by certain PTEN mutations in human cancers.     

Functional Interaction of Phosphatase and Tensin Homologue (PTEN) with the E3 Ligase NEDD4-1 during Neuronal Response to Zinc

The contribution of zinc-mediated neuronal death in the process of both acute and chronic neurodegeneration has been increasingly appreciated. Phosphatase and tensin homologue, deleted on chromosome 10 (PTEN), the major tumor suppressor and key regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, plays a critical role in neuronal death in response to various insults. NEDD4-1-mediated PTEN ubiquitination and subsequent degradation via the ubiquitin proteosomal system have recently been demonstrated to be the important regulatory mechanism for PTEN in several cancer types. We now demonstrate that PTEN is also the key mediator of the PI3K/Akt pathway in the neuronal response to zinc insult. We used primary cortical neurons and neuroblastoma N2a cells to show that zinc treatment results in a reduction of the PTEN protein level in parallel with increased NEDD4-1 gene/protein expression. The reduced PTEN level is associated with an activated PI3K pathway as determined by elevated phosphorylation of both Akt and GSK-3 as well as by the attenuating effect of a specific PI3K inhibitor (wortmannin). The reduction of PTEN can be attributed to increased protein degradation via the ubiquitin proteosomal system, as we show NEDD4-1 to be the major E3 ligase responsible for PTEN ubiquitination in neurons. Moreover, PTEN and NEDD4-1 appear to be able to counter-regulate each other to mediate the neuronal response to zinc. This reciprocal regulation requires the PI3K signaling pathway, suggesting a feedback loop mechanism. This study demonstrates that NEDD4-1-mediated PTEN ubiquitination is crucial in the regulation of PI3K/Akt signaling by PTEN during the neuronal response to zinc, which may represent a common mechanism in neurodegeneration.     

神经前体细胞表达发育性下调蛋白4在消化系统肿瘤发生中的作用

神经前体细胞表达发育性下调蛋白4(neural precursor cell expressed,developmentally down-regulated protein 4,NEDD4-1,部分文章也称NEDD4)是近年来才备受关注的肿瘤相关基因,属于E3 HECT(homologous to E6 associated protein C terminus,E6蛋白c端同源基因)泛素连接酶NEDD4样家族成员。泛素连接酶,能够参与多种蛋白质的泛素化、溶酶体及蛋白酶体的降解、胞核-胞质转位等,间接影响不同恶性肿瘤的多种信号通路。随着大量NEDD4-1与肿瘤相关实验的不断深入,目前已发现其可通过调控细胞周期、癌细胞侵袭转移、拮抗耐药性等许多途径影响肿瘤的生物学行为。在消化系统肿瘤中,NEDD4-1主要通过PTEN/PI3K/AKT、TGF-β、Hippo、LDLRAD4等多条通路促进肝细胞癌的增殖、侵袭和迁移能力;在胰腺癌中发现,NEDD4-1在PI3K/AKT信号通路中发挥癌基因作用,但在与Myc-SIRT2所形成的信号环路中,却发挥抑癌基因的作用;在胃癌和结直肠癌中,NEDD4-1所参与的信号通路与其他消化系统肿瘤均不相同,NEDD4-1能独立于PTEN/PI3K/AKT通路而发挥促进胃癌恶化、转移(EGFR信号通路)和抑制结直肠癌肿瘤生长(WNT信号通路)的作用。NEDD4-1已经成为人们治愈肿瘤的热门研究方向。本文通过系统总结NEDD4-1在不同消化系统肿瘤中的功能、信号通路和潜在抑制剂等,进行探讨NEDD4-1与不同信号通路的关系,旨为临床在癌症治疗领域提供重要的参考数据。     

Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.     

The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy

The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.     

The NEDD4-USP13 axis facilitates autophagy via deubiquitinating PIK3C3

ABSTRACT

Macroautophagy/autophagy, an evolutionarily conserved eukaryotic bioprocess, plays an important role in the bulk degradation of intracellular macromolecules, organelles, and invading pathogens. PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) functions as a key protein in autophagy initiation and progression. The activity of PIK3C3 is tightly regulated by multiple post-translational modifications, including ubiquitination, however, the regulatory mechanisms underpinning the reversible deubiquitination of PIK3C3 remain poorly understood. Recently, we identified the E3 ubiquitin ligase NEDD4/NEDD4-1 as a positive regulator of autophagy through decreasing the K48-linked ubiquitination of PIK3C3 by recruiting USP13.     

PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2)

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.     

NEDD4-1 Regulates Migration and Invasion of Glioma Cells through CNrasGEF Ubiquitination In Vitro

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) plays a great role in tumor cell growth, but its function and mechanism in cell invasive behavior are totally unknown. Here we report that NEDD4-1 regulates migration and invasion of malignant glioma cells via triggering ubiquitination of cyclic nucleotide Ras guanine nucleotide exchange factor (CNrasGEF) using cultured glioma cells. NEDD4-1 overexpression promoted cell migration and invasion, while its downregulation specifically inhibited them. However, NEDD4-1 did not affect the proliferation and apoptosis of glioma cells. NEDD4-1 physically interacted with CNrasGEF and promoted its poly-ubiquitination and degradation. Contrary to the effect of NEDD4-1, CNrasGEF downregulation promoted cell migration and invasion, while its overexpression inhibited them. Importantly, downregulation of CNrasGEF facilitated the effect of NEDD4-1-induced cell migration and invasion. Interestingly, aberrant up-regulated NEDD4-1 showed reverse correlation with CNrasGEF protein level but not with its mRNA level in glioma tissues. Combined with the in vitro results, the result of glioma tissues indicated post-translationally modification effect of NEDD4-1 on CNrasGEF. Our study suggests that NEDD4-1 regulates cell migration and invasion through ubiquitination of CNrasGEF in vitro.     

Upregulation of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation of PPAR-γ mediated by NEDD4-1

17β-estradiol (E2) treatment of cells results in an upregulation of SIRT1 and a down-regulation of PPARγ. The decrease in PPARγ expression is mediated by increased degradation of PPARγ. Here we report that PPARγ is ubiquitinated by HECT E3 ubiquitin ligase NEDD4-1 and degraded, along with PPARγ, in response to E2 stimulation. The PPARγ interacts with ubiquitin ligase NEDD4-1 through a conserved PPXY-WW binding motif. The WW3 domain in NEDD4-1 is critical for binding to PPARγ. NEDD4-1 overexpression leads to PPARγ ubiquitination and reduced expression of PPARγ. Conversely, knockdown of NEDD4-1 by specific siRNAs abolishes PPARγ ubiquitination. These data indicate that NEDD4-1 is the E3 ubiquitin ligase responsible for PPARγ ubiquitination. Here, we show that NEDD4-1 delays cellular senescence by degrading PPARγ expression. Taken together, our data show that E2 could upregulate SIRT1 expression via promoting the PPARγ ubiquitination-proteasome degradation pathway to delay the process of cell senescence.     

Aagab acts as a novel regulator of NEDD4-1-mediated Pten nuclear translocation to promote neurological recovery following hypoxic-ischemic brain damage

Hypoxic-ischemic encephalopathy (HIE) is a main cause of mortality and severe neurologic impairment in the perinatal and neonatal period. However, few satisfactory therapeutic strategies are available. Here, we reported that a rapid nuclear translocation of phosphatase and tensin homolog deleted on chromosome TEN (PTEN) is an essential step in hypoxic-ischemic brain damage (HIBD)- and oxygen-glucose deprivation (OGD)-induced neuronal injures both in vivo and in vitro. In addition, we found that OGD-induced nuclear translocation of PTEN is dependent on PTEN mono-ubiquitination at the lysine 13 residue (K13) that is mediated by neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). Importantly, we for the first time identified α- and γ-adaptin binding protein (Aagab) as a novel NEDD4-1 regulator to regulate the level of NEDD4-1, subsequently mediating Pten nuclear translocation. Finally, we demonstrated that genetic upregulation of Aagab or application of Tat-K13 peptide (a short interference peptide that flanks K13 residue of PTEN) not only reduced Pten nuclear translocation, but also significantly alleviated the deficits of myodynamia, motor and spatial learning and memory in HIBD model rats. These results suggest that Aagab may serve as a regulator of NEDD4-1-mediated Pten nuclear translocation to promote functional recovery following HIBD in neonatal rats, and provide a new potential therapeutic target to guide the clinical treatment for HIE.Subject terms: Cognitive ageing, Neurological disorders     

SIPL1-facilitated PTEN ubiquitination contributes to its association with PTEN

PTEN is post-translationally modified by ubiquitin via association with multiple E3 ubiquitin ligases, including NEDD4-1, XIAP, and WWP2. Despite the rapid progress made in researching the impact of ubiquitination on PTEN function, our understanding remains fragmented. Building on the previously observed interaction between SIPL1 and PTEN, we report here that SIPL1 promotes PTEN polyubiquitination via lysine 48 (K48)-independent polyubiquitin chains. Substitution of the K48 residue of ubiquitin with arginine (R) enhanced SIPL1-mediated PTEN polyubiquitination. In contrast, the K63R substitution significantly reduced it. The ubiquitin-like (UBL) domain is required for SIPL1-induced PTEN polyubiquitination. This post-translational modification promoted the association of SIPL1 with PTEN. Elevated amounts of the SIPL1/PTEN complex were precipitated in 293T cells co-transfected with PTEN, SIPL1, and ubiquitin compared to cells co-transfected with SIPL1 and PTEN only. Additionally, formation of the SIPL1/PTEN complex was inhibited when either lysine-less (K0) ubiquitin or K63R ubiquitin was co-transfected together with SIPL1 + PTEN. The PTEN component in the SIPL1/PTEN complex contained polyubiquitin chains. The ubiquitination reaction may play a structural role, stabilizing the SIPL1/PTEN complex, as a ubiquitin binding-defective SIPL1 mutant (TFLV) is proficient in PTEN association. Collectively, we demonstrate that SIPL1 binds PTEN and enhances PTEN polyubiquitination which in turn promotes the interaction between SIPL1 and PTEN.     

RFP-mediated ubiquitination of PTEN modulates its effect on AKT activation

The PTEN tumor suppressor is a lipid phosphatase that has a central role in regulating the phosphatidylinositol-3-kinase (PI3K) signal transduction cascade. Nevertheless, the mechanism by which the PTEN activity is regulated in cells needs further elucidation. Although previous studies have shown that ubiquitination of PTEN can modulate its stability and subcellular localization, the role of ubiquitination in the most critical aspect of PTEN function, its phosphatase activity, has not been fully addressed. Here, we identify a novel E3 ubiquitin ligase of PTEN, Ret finger protein (RFP), that is able to promote atypical polyubiquitinations of PTEN. These ubiquitinations do not lead to PTEN instability or relocalization, but rather significantly inhibit PTEN phosphatase activity and therefore modulate its ability to regulate the PI3K signal transduction cascade. Indeed, RFP overexpression relieves PTEN-mediated inhibitory effects on AKT activation; in contrast, RNAi-mediated knockdown of endogenous RFP enhances the ability of PTEN to suppress AKT activation. Moreover, RFP-mediated ubiquitination of PTEN inhibits PTEN-dependent activation of TRAIL expression and also suppresses its ability to induce apoptosis. Our findings demonstrate a crucial role of RFP-mediated ubiquitination in controlling PTEN activity.     

MGRN1‐dependent pigment‐type switching requires its ubiquitination activity but not its interaction with TSG101 or NEDD4

Mice lacking the E3 ubiquitin ligase mahogunin ring finger‐1 (MGRN1) have a pleiotropic phenotype that includes spongiform neurodegeneration, embryonic patterning defects, and dark fur due to a defect in pigment‐type switching. The only MGRN1 ubiquitination target identified to date is tumor susceptibility gene 101 (TSG101), a component of the endosomal trafficking machinery. Here, we show that MGRN1 also interacts with but does not ubiquitinate NEDD4, a HECT‐domain ubiquitin ligase involved in endosomal trafficking. Using transgenesis in mice, we demonstrate that pigment‐type switching likely requires MGRN1′s ubiquitin ligase activity but not its ability to bind TSG101 or NEDD4. This indicates that MGRN1‐dependent ubiquitination of an as‐yet unidentified target protein is required for agouti‐mediated melanocortin signaling.