E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases.     

Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway

The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4^DCAF1. Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin''s auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin''s binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin''s auto-inhibitory tail does not obviously alter Merlin''s conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.     

NEDD4 attenuates phosgene‐induced acute lung injury through the inhibition of Notch1 activation

Phosgene gas leakage can cause life‐threatening acute lung injury (ALI), which is characterized by inflammation, increased vascular permeability, pulmonary oedema and oxidative stress. Although the downregulation of neuronal precursor cell‐expressed developmentally downregulated 4 (NEDD4) is known to be associated with inflammation and oxidative damage, its functions in phosgene‐induced ALI remain unclear. In this study, rats with phosgene‐induced ALI were intravenously injected with NEDD4‐overexpressing lentiviruses to determine the functions of NEDD4 in this inflammatory condition. NEDD4 expression was decreased in the lung parenchyma of phosgene‐exposed control rats, whereas its expression level was high in the NEDD4‐overexpressing rats. Phosgene exposure increased the wet‐to‐dry lung weight ratio, but NEDD4 abrogated this effect. NEDD4 overexpression attenuated phosgene‐induced lung inflammation, lowering the high lung injury score (based on total protein, inflammatory cells and inflammatory factors in bronchoalveolar lavage fluid) and also reduced phosgene‐induced oxidative stress and cell apoptosis. Finally, NEDD4 was found to interact with Notch1, enhancing its ubiquitination and thereby its degradation, thus attenuating the inflammatory responses to ALI. Therefore, we demonstrated that NEDD4 plays a protective role in alleviating phosgene‐induced ALI, suggesting that enhancing the effect of NEDD4 may be a new approach for treating phosgene‐induced ALI.     

E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation

Psoriasis is mainly characterized by abnormal hyperplasia of keratinocytes and immune cells infiltrating into the dermis and epidermis. Neural precursor cell expressed developmentally downregulated 4‐like (NEDD4L) is a highly conserved HECT type E3 ligase that plays an important role in regulating physiological and pathological processes. Here, we identify NEDD4L as a negative regulator of psoriasis. Nedd4l significantly inhibits imiquimod (IMQ)‐induced skin hyperplasia, and this effect is attributed to the inhibitory effect of NEDD4L on IL‐6/GP130 signaling in keratinocytes. Mechanistically, NEDD4L directly interacts with GP130 and mediates its Lys‐27‐linked ubiquitination and proteasomal degradation. Moreover, the expression of NEDD4L is downregulated in the epidermis from IMQ‐treated mice and psoriasis patients and negatively correlates with the protein levels of GP130 and p‐STAT3 in clinical samples. Collectively, we uncover an inhibitory role of NEDD4L in the pathogenesis of psoriasis and suggest a new therapeutic strategy for the treatment of psoriasis.     

p34 is a novel regulator of the oncogenic behavior of NEDD4-1 and PTEN

PTEN is one of the most frequently mutated or deleted tumor suppressors in human cancers. NEDD4-1 was recently identified as the E3 ubiquitin ligase for PTEN; however, a number of important questions remain regarding the role of ubiquitination in regulating PTEN function and the mechanisms by which PTEN ubiquitination is regulated. In the present study, we demonstrated that p34, which was identified as a binding partner of NEDD4-1, controls PTEN ubiquitination by regulating NEDD4-1 protein stability. p34 interacts with the WW1 domain of NEDD4-1, an interaction that enhances NEDD4-1 stability. Expression of p34 promotes PTEN poly-ubiquitination, leading to PTEN protein degradation, whereas p34 knockdown results in PTEN mono-ubiquitination. Notably, an inverse correlation between PTEN and p34/NEDD4-1 levels was confirmed in tumor samples from colon cancer patients. Thus, p34 acts as a key regulator of the oncogenic behavior of NEDD4-1 and PTEN.     

Multiphase coalescence mediates Hippo pathway activation

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NEDD4‐induced degradative ubiquitination of phosphatidylinositol 4‐phosphate 5‐kinase α and its implication in breast cancer cell proliferation

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) family members generate phosphatidylinositol 4,5‐bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K‐dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin‐proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down‐regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C‐terminal region and ubiquitinated the N‐terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)‐induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4‐mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild‐type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα‐dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.     

Arrestin domain‐containing protein 3 recruits the NEDD4 E3 ligase to mediate ubiquitination of the β2‐adrenergic receptor

Prolonged stimulation of the β2‐adrenergic receptor (β2AR) leads to receptor ubiquitination and downregulation. Using a genome‐wide RNA interference screen, we identified arrestin domain‐containing 3 (ARRDC3) as a gene required for β2AR regulation. The ARRDC3 protein interacts with ubiquitin ligase neural precursor development downregulated protein 4 (NEDD4) through two conserved PPXY motifs and recruits NEDD4 to the activated receptor. The ARRDC3 protein also interacts and co‐localizes with activated β2AR. Knockdown of ARRDC3 expression abolishes the association between NEDD4 and β2AR. Furthermore, functional inactivation of ARRDC3, either through small interfering RNA (siRNA)‐mediated knockdown or overexpression of a mutant that does not interact with NEDD4, blocks receptor ubiquitination and degradation. Our results establish ARRDC3 as an essential adaptor for β2AR ubiquitination.     

WW domain interactions regulate the Hippo tumor suppressor pathway

The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis in Drosophila and mammalians. Although the signaling of the core kinases is relatively well understood, less is known about the upstream inputs, downstream outputs and regulation of the whole cascade. Enrichment of the Hippo pathway components with WW domains and their cognate proline-rich interacting motifs provides a versatile platform for further understanding the mechanisms that regulate organ growth and tumorigenesis. Here, we review recently discovered mechanisms of WW domain-mediated interactions that contribute to the regulation of the Hippo signaling pathway in tumorigenesis. We further discuss new insights and future directions on the emerging role of such regulation.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

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.     

hCINAP regulates the differentiation of embryonic stem cells by regulating NEDD4 liquid-liquid phase-separation-mediated YAP1 activation

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The CTLA‐4 immune checkpoint protein regulates PD‐L1:PD‐1 interaction via transendocytosis of its ligand CD80

CTLA‐4 and PD‐1 are key immune checkpoint receptors that are targeted in the treatment of cancer. A recently identified physical interaction between the respective ligands, CD80 and PD‐L1, has been shown to block PD‐L1/PD‐1 binding and to prevent PD‐L1 inhibitory functions. Since CTLA‐4 is known to capture and degrade its ligands via transendocytosis, we investigated the interplay between CD80 transendocytosis and CD80/PD‐L1 interaction. We find that transendocytosis of CD80 results in a time‐dependent recovery of PD‐L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is internalised, while its heterodimeric PD‐L1 partner remains on the plasma membrane of the antigen‐presenting cell (APC). CTLA‐4 interactions with CD80 do not appear to be inhibited by PD‐L1, but efficient removal of CD80 requires an intact CTLA‐4 cytoplasmic domain, distinguishing this process from more general trogocytosis and simple CTLA‐4 binding to CD80/PD‐L1 complexes. These data are consistent with CTLA‐4 acting as modulator of PD‐L1:PD‐1 interactions via control of CD80.     

ANKHD1, a novel component of the Hippo signaling pathway,promotes YAP1 activation and cell cycle progression in prostate cancer cells

ANKHD1 is a multiple ankyrin repeat containing protein, recently identified as a novel member of the Hippo signaling pathway. The present study aimed to investigate the role of ANKHD1 in DU145 and LNCaP prostate cancer cells. ANKHD1 and YAP1 were found to be highly expressed in prostate cancer cells, and ANKHD1 silencing decreased cell growth, delayed cell cycle progression at the S phase, and reduced tumor xenograft growth. Moreover, ANKHD1 knockdown downregulated YAP1 expression and activation, and reduced the expression of CCNA2, a YAP1 target gene. These findings indicate that ANKHD1 is a positive regulator of YAP1 and promotes cell growth and cell cycle progression through Cyclin A upregulation.     

Long non‐coding RNA TNRC6C‐AS1 promotes methylation of STK4 to inhibit thyroid carcinoma cell apoptosis and autophagy via Hippo signalling pathway

The role of long non‐coding RNAs (lncRNAs) in thyroid carcinoma (TC), the most frequent endocrine malignancy, has been extensively examined. This study investigated effect of interaction among lncRNA TNRC6C‐AS1, serine/threonine‐protein kinase 4 (STK4) and Hippo signalling pathway on TC. Initially, lncRNA TNRC6C‐AS1 expression in TC tissues was detected. To explore roles of lncRNA TNRC6C‐AS1, STK4 and Hippo signalling pathway in TC progression, their expressions were altered. Interaction between lncRNA TNRC6C‐AS1 and STK4, STK4 promoter methylation, or Hippo signalling pathway was verified. After that, a series of experiments were employed to evaluate in vitro ability of apoptosis, proliferation and autophagy of TC cells and in vivo tumorigenicity, and tumour growth of TC cells. lncRNA TNRC6C‐AS1 was highly expressed while STK4 was poorly expressed in TC tissues. LncRNA TNRC6C‐AS1 promoted the STK4 methylation and down‐regulated STK4 expression, which further activated the Hippo signalling pathway. STK4 silencing was observed to promote the proliferation ability of TC cells, inhibit the apoptosis and autophagy abilities, as well as enhance the tumorigenicity and tumour growth. Moreover, the in vitro proliferation ability as well as the in vivo tumorigenicity and tumour growth of TC cells were inhibited after the blockade of Hippo signalling pathway, while the apoptosis and autophagy abilities were promoted. The results demonstrate that the lncRNA TNRC6C‐AS1 increases STK4 promoter methylation to down‐regulate STK4 expression, thereby promoting the development of TC through activation of Hippo signalling pathway. It highlights that lncRNA TNRC6C‐AS1 may be a novel therapeutic target for the treatment of TC.     

BCAS3 exhibits oncogenic properties by promoting CRL4A‐mediated ubiquitination of p53 in breast cancer

ObjectivesBreast cancer‐amplified sequence 3 (BCAS3) was initially found to be amplified in human breast cancer (BRCA); however, there has been little consensus on the functions of BCAS3 in breast tumours.Materials and methodsWe analysed BCAS3 expression in BRCA using bio‐information tools. Affinity purification and mass spectrometry were employed to identify BCAS3‐associated proteins. GST pull‐down and ubiquitination assays were performed to analyse the interaction mechanism between BCAS3/p53 and CUL4A‐RING E3 ubiquitin ligase (CRL4A) complex. BCAS3 was knocked down individually or in combination with p53 in MCF‐7 cells to further explore the biological functions of the BCAS3/p53 axis. The clinical values of BCAS3 for BRCA progression were evaluated via semiquantitative immunohistochemistry (IHC) analysis and Cox regression.ResultsWe reported that the expression level of BCAS3 in BRCA was higher than that in adjacent normal tissues. High BCAS3 expression promoted growth, inhibited apoptosis and conferred chemoresistance in breast cancer cells. Mechanistically, BCAS3 overexpression fostered BRCA cell growth by interacting with the CRL4A complex and promoting ubiquitination and proteasomal degradation of p53. Furthermore, BCAS3 could regulate cell growth, apoptosis and chemoresistance through a p53‐mediated mechanism. Clinically, BCAS3 overexpression was significantly correlated with a malignant phenotype. Moreover, higher expression of BCAS3 correlates with shorter overall survival (OS) in BRCA.ConclusionsThe functional characterization of BCAS3 offers new insights into the oncogenic properties and chemotherapy resistance in breast cancer.