The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC-RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.     

Membrane-tethered Notch1 exhibits oncogenic property via activation of EGFR–PI3K–AKT pathway in oral squamous cell carcinoma

Notch proteins are highly conserved cell surface receptors which play essential roles in cellular differentiation, proliferation, and apoptotic events at all stages of development. Recently, NOTCH1 mutations have been extensively observed in oral squamous cell carcinoma (OSCC) and are hinted to be Notch1-inactivating mutations. However, little is known about the biological effect of these reported mutations in OSCC. To mimic the inactivation of Notch1 due to inappropriate mutations and to determine the potential mechanisms, we utilized wild-type Notch1 vectors (Notch1^WT) or mutant Notch1 vectors (Notch1^V1754L) to transfect into OSCC cell lines. Membrane-tethered Notch1 induced by mutation was analyzed by immunofluorescence staining. γ-Secretase inhibitor PF-03084014 was utilized to determine the phenotype in the absence of endogenous Notch1 activation. Here we demonstrated that membrane-tethered Notch1 inactivated the canonical Notch1 signaling and oncogenic phenotypes were identified by promoting cell proliferation and invasion and by inducing epithelial-to-mesenchymal transition in cells. The γ-secretase inhibitor PF-03084014 also showed distinct oncogenic property after treatment. Importantly, both membrane-tethered Notch1 and PF-03084014 inhibitor activated the epidermal growth factor receptor (EGFR)–phosphoinositide 3-kinase (PI3K)–protein kinase B (AKT) signaling pathway, which has been confirmed as an overwhelming modulator in OSCC. This was the first time that we clearly simulated the mutated Notch1 activities and determined the oncogenic phenotypes of membrane-tethered Notch1. Compared with wild-type Notch1, membrane-tethered Notch1 was strongly associated with activated EGFR–PI3K–AKT signaling pathway.     

Adropin Is a Brain Membrane-bound Protein Regulating Physical Activity via the NB-3/Notch Signaling Pathway in Mice

Adropin is a highly conserved polypeptide that has been suggested to act as an endocrine factor that plays important roles in metabolic regulation, insulin sensitivity, and endothelial functions. However, in this study, we provide evidence demonstrating that adropin is a plasma membrane protein expressed abundantly in the brain. Using a yeast two-hybrid screening approach, we identified NB-3/Contactin 6, a brain-specific, non-canonical, membrane-tethered Notch1 ligand, as an interaction partner of adropin. Furthermore, this interaction promotes NB3-induced activation of Notch signaling and the expression of Notch target genes. We also generated and characterized adropin knockout mice to explore the role of adropin in vivo. Adropin knockout mice exhibited decreased locomotor activity and impaired motor coordination coupled with defective synapse formation, a phenotype similar to NB-3 knockout mice. Taken together, our data suggest that adropin is a membrane-bound protein that interacts with the brain-specific Notch1 ligand NB3. It regulates physical activity and motor coordination via the NB-3/Notch signaling pathway and plays an important role in cerebellum development in mice.     

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP-Jkappa/CBF-1 and GC-rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1-induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.     

Atrophin-1-interacting protein 4/human Itch is a ubiquitin E3 ligase for human enhancer of filamentation 1 in transforming growth factor-beta signaling pathways

Atrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-beta (TGF-beta) signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-beta type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-beta stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. Here we report our studies that demonstrate the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-beta-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-beta stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-beta signaling pathway and those involving HEF1 and AIP4.     

A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex

The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.     

The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.     

Notch3 Interactome Analysis Identified WWP2 as a Negative Regulator of Notch3 Signaling in Ovarian Cancer

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.     

Xenopus neuralized is a ubiquitin ligase that interacts with XDelta1 and regulates Notch signaling.

Notch signaling in Drosophila requires a RING finger (RF) protein encoded by neuralized. Here we show that the Xenopus homolog of neuralized (Xneur) is expressed where Notch signaling controls cell fate choices in early embryos. Overexpressing XNeur or putative dominant-negative forms in embryos inhibits Notch signaling. As expected for a RF protein, we show that XNeur fulfills the biochemical requirements of ubiquitin ligases. We also show that wild-type XNeur decreases the cell surface level of the Notch ligand, XDelta1, while putative inhibitory forms of XNeur increase it. Finally, we provide evidence that XNeur acts as a ubiquitin ligase for XDelta1 in vitro. We propose that XNeur plays a conserved role in Notch activation by regulating the cell surface levels of the Delta ligands, perhaps directly, via ubiquitination.     

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.Notch signaling determines cell fate and affects cell proliferation, differentiation, and apoptosis during cell development (1). As a highly conserved family, Notch coordinates a signaling cascade present in all animal species studied to date (2). Mammals have four Notch receptors that bind five different ligands, among which Notch1 signaling functions in many physiological and pathophysiological processes of numerous cell types, and its dysfunction results in a variety of developmental defects, including embryonic lethality and adult disorders. For example, the Notch1/Jagged1 signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting hepatocyte growth (3, 4). Inducible inactivation of Notch1 has been shown to cause nodular regenerative hyperplasia in mouse liver (5). These studies suggest that Notch1 signaling may be involved in the liver functions and the pathogenesis of liver diseases. Our previous study demonstrated that Notch1 signaling could suppress the growth of human hepatocellular carcinoma (HCC)⁴ cells by arresting the cell cycle and inducing apoptosis (6). However, the underlying molecular mechanisms remain to be fully understood.p53, an important tumor suppressor gene, is involved in cell cycle arrest and cellular apoptosis. Its activity is mostly regulated by complex networks of post-translational modifications, including phosphorylation, ubiquitination, and proteasome degradation. One protein that is essential for determining p53 stability is Mdm2 (mouse double minute protein 2) (7). Mdm2, a nuclear phosphoprotein and an E3 ubiquitin ligase, binds to p53 and ubiquitinates p53, leading to proteosome degradation of p53 (8). Another important mechanism of p53 stability is related to its phosphorylation status, which is Mdm2-dependent or Mdm2-independent (9). As to the regulation of p53 by Notch1, there are controversial reports that Notch1 activation increased p53 expression in neural progenitor cells (10); however, suppression of p53 by Notch signaling was also well established in lymphomagenesis (11). We also reported that Notch1 signaling significantly up-regulated p53 expression in SMMC7721 HCC cells (6); however, the molecular mechanisms remained unclear and needed to be further characterized.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of a superfamily of cell death-inducing ligands, induces apoptosis in a broad range of transformed cells and tumor cells but has little or no effect on normal cells (12). Therefore, TRAIL has been regarded as a potential drug for cancer therapy (12, 13). However, several kinds of cancer, including HCC, are not sensitive to soluble TRAIL treatment (14). HCC accounts for 80–90% of liver cancers and is one of the most prevalent carcinomas throughout the world, especially in Africa and Asia. Thus, it is worthwhile to find a new strategy to overcome the resistance of HCC cells to TRAIL-induced apoptosis.Considering that Notch1 signaling up-regulates p53 and induces apoptosis of HCC cells and that there are no reports to date that address the relationship between Notch1 signaling and TRAIL-induced apoptosis, in this study, we investigated whether and how Notch1 signaling could sensitize HCC cells to TRAIL-induced apoptosis. We demonstrate that Notch1 signaling up-regulates p53 expression by inhibiting proteasome degradation via, at least in part, suppressing the phosphatidylinositol 3-kinase/Akt/Hdm2 pathway. In addition, we here report that Notch1 signaling enhances DR5 (death receptor 5) expression in a p53-dependent manner, and DR5 contributes, at least in part, to the enhancement of TRAIL-induced apoptosis by Notch1 signaling. Accordingly, Notch1 signaling sensitizes HCC cells to TRAIL-induced apoptosis.