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Notch信号通路与多种肿瘤的发生发展密切相关。对该通路中各受体功能的深入研究能够为揭示其致癌作用奠定基础。本研究采用RT-PCR的方法从人宫颈癌细胞系HeLa细胞的cDNA中克隆人Notch2和Notch3受体胞内区基因(N2ICD和N3ICD基因),并构建携带增强型绿色荧光蛋白(EGFP)的真核表达载体N2ICD/pEGFP和N3ICD/pEGFP,将序列正确的重组质粒转染HeLa细胞,显微镜下可见明显的绿色荧光,并定位在细胞核内。Western blotting检测目的蛋白成功融合表达,并能够提高其下游靶基因Hes1的转录活性。 相似文献
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Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation 总被引:11,自引:0,他引:11
Dahlqvist C Blokzijl A Chapman G Falk A Dannaeus K Ibâñez CF Lendahl U 《Development (Cambridge, England)》2003,130(24):6089-6099
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Calcium depletion dissociates and activates heterodimeric notch receptors 总被引:22,自引:0,他引:22
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Rand MD Grimm LM Artavanis-Tsakonas S Patriub V Blacklow SC Sklar J Aster JC 《Molecular and cellular biology》2000,20(5):1825-1835
Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit. 相似文献
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Nakhai H Siveke JT Klein B Mendoza-Torres L Mazur PK Algül H Radtke F Strobl L Zimber-Strobl U Schmid RM 《Development (Cambridge, England)》2008,135(16):2757-2765
The role of the Notch signaling members Notch1, Notch2 and Rbpj in exocrine pancreatic development is not well defined. We therefore analyzed conditional pancreas-specific Rbpj and combined Notch1/Notch2 knockout mice using Ptf1a(+/Cre(ex1)) mice crossed with floxed Rbpj or Notch1/Notch2 mice. Mice were analyzed at different embryonic stages for pancreatic exocrine and endocrine development. The absence of Rbpj in pancreatic progenitor cells impaired exocrine pancreas development up to embryonic day 18.5 and led to premature differentiation of pancreatic progenitors into endocrine cells. In Rbpj-deficient pancreata, amylase-expressing acini and islets formed during late embryonic and postnatal development, suggesting an essential role of Rbpj in early but not late development. Contrary to this severe phenotype, the concomitant inactivation of Notch1 and Notch2 only moderately disturbed the proliferation of pancreatic epithelial cells during early embryonic development, and did not inhibit pancreatic development. Our results show that, in contrast to Rbpj, Notch1 and Notch2 are not essential for pancreatogenesis. These data favor a Notch-independent role of Rbpj in the development of the exocrine pancreas. Furthermore, our findings suggest that in late stages of pancreatic development exocrine cell differentiation and maintenance are independent of Rbpj. 相似文献
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Krivtsov AV Rozov FN Zinovyeva MV Hendrikx PJ Jiang Y Visser JW Belyavsky AV 《Journal of cellular biochemistry》2007,101(3):767-784
Self-renewal and differentiation of hematopoietic stem and progenitor cells are defined by the ensembles of genes expressed by these cells. Here we report identification of a novel gene named Jedi, which is expressed predominantly in short- and long-term repopulating stem cells when compared to more mature bone marrow progenitors. Jedi mRNA encodes a transmembrane protein that contains multiple EGF-like repeats. Jedi and two earlier reported proteins, MEGF10 and MEGF11, share a substantial homology and are likely to represent a novel protein family. Studies of the potential role of Jedi in hematopoietic regulation demonstrated that the retrovirally mediated expression of Jedi in bone marrow cells decreased the number of myeloid progenitors in in vitro clonogenic assays. In addition, expression of Jedi in NIH 3T3 fibroblasts resulted in a decreased number of late and early myeloid progenitors in the non-adherent co-cultured bone marrow cells. Jedi shares a number of structural features with the Jagged/Serrate/Delta family of Notch ligands, and our experiments indicate that the extracellular domain of Jedi, similar to the corresponding domain of Jagged1, inhibits Notch signaling. On the basis of obtained results, we suggest that Jedi is involved in the fine regulation of the early stages of hematopoietic differentiation, presumably through the Notch signaling pathway. 相似文献
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SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation
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Wu G Lyapina S Das I Li J Gurney M Pauley A Chui I Deshaies RJ Kitajewski J 《Molecular and cellular biology》2001,21(21):7403-7415
Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ. 相似文献
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RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1
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Daniel Hall Francesca Ferrante Diana M Ho Kazuya Hori Lucas Anhezini Iris Ertl Marek Bartkuhn Honglai Zhang Eléna Milon Kimberly Ha Kevin P Conlon Rork Kuick Brandon Govindarajoo Yang Zhang Yuqing Sun Yali Dou Venkatesha Basrur Kojo SJ Elenitoba‐Johnson Alexey I Nesvizhskii Julian Ceron Cheng‐Yu Lee Tilman Borggrefe Rhett A Kovall Jean‐François Rual 《The EMBO journal》2017,36(21):3232-3249
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Lijun Jia Genggeng Yu Yanmei Zhang Michael M. Wang 《The international journal of biochemistry & cell biology》2009,41(12):2594-2598
Notch signaling plays an essential role in diverse biological processes during development and in pathogenesis of diseases ranging from cancer to cerebrovascular disorders. Precise regulation of Notch signaling is essential for normal function and requires both timely activation and inactivation of the intracellular domain (ICD) of Notch receptors. In addition, inappropriate buildup of Notch3 ectodomain is a hallmark pathological feature of the stroke and dementia disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Thus, a clear understanding of mechanisms of Notch protein turnover is essential for understanding normal and pathological mechanisms of Notch function. Previous studies showed that the degradation of ICDs of Notch1 and Notch4 is controlled by the ubiquitin–proteasome system (UPS), though more recent work demonstrated that Notch1 ICD is also controlled by lysosomal degradation. The mechanism of degradation of Notch3 has not yet been identified. Here we report that the degradation of ICD of Notch3 (N3-ICD) is mediated by lysosomes. Lysosome inhibitors chloroquine and NH4Cl led to the accumulation of transfected N3-ICD in 293 cells and endogenous N3-ICD in C2C12, H460, and HeLa cell lines; in addition, inhibition of lysosome function by chloroquine and NH4Cl delayed the degradation of N3-ICD. In contrast, N3-ICD was not affected by proteasome inhibitors MG132 and lactacystin. Furthermore, we find that the Notch3 extracellular domain (N3-ECD) is also subjected to lysosome-dependent degradation. In sum, our experiments demonstrate a critical role for lysosomes in the degradation of Notch3, which distinguishes it from Notch1 and Notch4. 相似文献
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Sakamoto K Yamaguchi S Ando R Miyawaki A Kabasawa Y Takagi M Li CL Perbal B Katsube K 《The Journal of biological chemistry》2002,277(33):29399-29405
We demonstrate a novel interaction of the nephroblastoma overexpressed gene (NOV), a member of the CCN gene family, with the Notch signaling pathway. NOV associates with the epidermal growth factor-like repeats of Notch1 by the CT (C-terminal cysteine knot) domain. The promoters of HES1 and HES5, which are the downstream transducers of Notch signaling, were activated by NOV. Expressions of NOV and Notch1 were concomitant in the presomitic mesoderm and later in the myocytes and chondrocytes, suggesting their synergistic effects in mesenchymal cell differentiation. In C2/4 myogenic cells, elevated expression of NOV led to down-regulation of MyoD and myogenin, resulting in inhibition of myotube formation. These results indicate that NOV-Notch1 association exerts a positive effect on Notch signaling and consequently suppresses myogenesis. 相似文献
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Marie Blanke Andrawes Xiang Xu Hong Liu Scott B. Ficarro Jarrod A. Marto Jon C. Aster Stephen C. Blacklow 《The Journal of biological chemistry》2013,288(35):25477-25489
Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6–15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8–10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6–15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor-mediated responses of developmental signaling pathways. 相似文献
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Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1
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Yang LT Nichols JT Yao C Manilay JO Robey EA Weinmaster G 《Molecular biology of the cell》2005,16(2):927-942
Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events. 相似文献
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