NUMB enhances Notch signaling by repressing ubiquitination of NOTCH1 intracellular domain

Abstract The release and nuclear translocation of the intracellular domain of Notch receptor (NICD) is the prerequisite for Notch signaling-mediated transcriptional activation. NICD is subjected to various posttranslational modifications including ubiquitination. Here, we surprisingly found that NUMB proteins stabilize the intracellular domain of NOTCH1 receptor (N1ICD) by regulating the ubiquitin–proteasome machinery, which is independent of NUMB’s role in modulating endocytosis. BAP1, a deubiquitinating enzyme (DUB), was further identified as a positive N1ICD regulator, and NUMB facilitates the association between N1ICD and BAP1 to stabilize N1ICD. Intriguingly, BAP1 stabilizes N1ICD independent of its DUB activity but relying on the BRCA1-inhibiting function. BAP1 strengthens Notch signaling and maintains stem-like properties of cortical neural progenitor cells. Thus, NUMB enhances Notch signaling by regulating the ubiquitinating activity of the BAP1–BRCA1 complex.

Download Full-text

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

Science Signaling ◽

10.1126/scisignal.aay2369 ◽

2019 ◽

Vol 12 (606) ◽

pp. eaay2369 ◽

Cited By ~ 3

Author(s):

Sanchez M. Jarrett ◽

Tom C. M. Seegar ◽

Mark Andrews ◽

Guillaume Adelmant ◽

Jarrod A. Marto ◽

...

Keyword(s):

Notch Signaling ◽

Negative Feedback ◽

Transcriptional Activation ◽

Cultured Cells ◽

Feedback Inhibition ◽

Lymphoblastic Leukemia ◽

Ankyrin Repeat ◽

Structural Basis ◽

Intracellular Domain ◽

Notch Intracellular Domain

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

Download Full-text

Tax ubiquitylation and sumoylation control critical cytoplasmic and nuclear steps of NF-κB activation

Blood ◽

10.1182/blood-2005-09-3572 ◽

2006 ◽

Vol 107 (10) ◽

pp. 4021-4029 ◽

Cited By ~ 70

Author(s):

Rihab Nasr ◽

Estelle Chiari ◽

Marwan El-Sabban ◽

Renaud Mahieux ◽

Youmna Kfoury ◽

...

Keyword(s):

Posttranslational Modifications ◽

Transcriptional Activation ◽

Nuclear Translocation ◽

Leukemia Virus ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Nuclear Bodies ◽

Type I ◽

Cell Leukemia Virus Type ◽

Human T Cell

The Tax oncoprotein plays a crucial role in the proliferation and transformation of human T-cell leukemia virus type I (HTLV-I)–infected T lymphocytes through various mechanisms, including activation of the nuclear factor (NF)–κB pathway. We found that cytoplasmic ubiquitylation of Tax C-terminal lysines is critical for Tax binding to the IkappaB kinase complex and subsequent nuclear translocation of RelA. Conversely, we demonstrate that the same lysines are sumoylated in the nucleus, an event required for the formation of RelA/p300-enriched Tax nuclear bodies and full NF-κB transcriptional activation. In contrast, Tax ubiquitylation and sumoylation are dispensable for its activation of cyclic adenosine monophosphate response element binding protein (CREB)–dependent genes. Thus, ubiquitylation and sumoylation of the same residues of Tax regulate 2 essential steps controlling NF-κB activation, demonstrating how these posttranslational modifications can cooperate to promote Tax-induced transformation.

Download Full-text

SEL-10 Is an Inhibitor of Notch Signaling That Targets Notch for Ubiquitin-Mediated Protein Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7403-7415.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7403-7415 ◽

Cited By ~ 226

Author(s):

Guangyu Wu ◽

Svetlana Lyapina ◽

Indranil Das ◽

Jinhe Li ◽

Mark Gurney ◽

...

Keyword(s):

Notch Signaling ◽

Protein Degradation ◽

Notch Receptor ◽

Dominant Negative ◽

Intracellular Domain ◽

Notch Receptors ◽

Intracellular Domains ◽

Wd40 Repeats ◽

In Cells

ABSTRACT 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.

Download Full-text

The Neogenin Intracellular Domain Regulates Gene Transcription via Nuclear Translocation

Molecular and Cellular Biology ◽

10.1128/mcb.02114-07 ◽

2008 ◽

Vol 28 (12) ◽

pp. 4068-4079 ◽

Cited By ~ 35

Author(s):

David Goldschneider ◽

Nicolas Rama ◽

Catherine Guix ◽

Patrick Mehlen

Keyword(s):

Cell Death ◽

Transcriptional Regulation ◽

Neuronal Differentiation ◽

Nuclear Import ◽

Nuclear Translocation ◽

Notch Receptor ◽

Nuclear Proteins ◽

Nuclear Chromatin ◽

Intracellular Domain ◽

Import And Export

ABSTRACT Neogenin is a multifunctional receptor implicated in axon navigation, neuronal differentiation, morphogenesis, and cell death. Very little is known about signaling downstream of neogenin. Because we found that the neogenin intracellular domain (NeICD) interacts with nuclear proteins implicated in transcription regulation, we investigated further whether neogenin signals similarly to the Notch receptor. We show here that neogenin is cleaved by γ-secretase, an event that releases the complete NeICD. We also describe that NeICD is located at the nucleus, a feature regulated through a balance between nuclear import and export. NeICD triggers gene reporter transactivation and associates with nuclear chromatin. Direct transcriptional targets of NeICD were determined and were shown to be up-regulated in the presence of neogenin ligand. Together, we reveal here a novel aspect of neogenin signaling that relies on the direct implication of its intracellular domain in transcriptional regulation.

Download Full-text

Modulation of p53 Degradation via MDM2-Mediated Ubiquitylation and the Ubiquitin–Proteasome System during Reperfusion after Stroke: Role of Oxidative Stress

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600028 ◽

2005 ◽

Vol 25 (2) ◽

pp. 267-280 ◽

Cited By ~ 33

Author(s):

Atsushi Saito ◽

Takeshi Hayashi ◽

Shuzo Okuno ◽

Tatsuro Nishi ◽

Pak H Chan

Keyword(s):

Cerebral Ischemia ◽

Transcriptional Activation ◽

Nuclear Translocation ◽

Focal Cerebral Ischemia ◽

Cell Cycle Control ◽

Ubiquitin Proteasome System ◽

Suppressor Gene ◽

Copper Zinc Superoxide Dismutase ◽

Ubiquitin Proteasome ◽

Copper Zinc

The tumor suppressor gene p53 plays an important role in the regulation of apoptosis through transcriptional activation of cell cycle control. Degradation of p53 hinders its role in apoptosis regulation. Recent studies have shown that MDM2-mediated ubiquitylation and the ubiquitin–proteasome system are critical regulating systems of p53 ubiquitylation. However, the mechanism regulating p53-mediated neuronal apoptosis after cerebral ischemia remains unknown. We examined the MDM2 pathway and the ubiquitin–proteasome system using a transient focal cerebral ischemia (tFCI) model and analyzed the interaction between p53 regulation and superoxide using copper/zinc superoxide dismutase (SOD1) transgenic mice after tFCI. p53 degradation and ubiquitylation were detected after tFCI. The accumulation of ubiquitylated p53 was inhibited and p53 degradation was facilitated by SOD1. Nuclear translocation and MDM2/Akt interaction were detected after tFCI and were inhibited by phosphatidylinositol 3-kinase inhibition and promoted by SOD1. Cytosolic translocation of the p53/MDM2 complex was detected after tFCI and was promoted by SOD1. Moreover, accumulation of multiubiquitin chains and direct oxidative injury to a proteasome were detected and inhibited by SOD1 after tFCI. These results suggest that SOD1 promotes the MDM2 pathway and the ubiquitin–proteasome system after tFCI and that production of reactive oxygen species after tFCI prevents p53 degradation by inhibiting both systems.

Download Full-text

Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification

Molecular and Cellular Biology ◽

10.1128/mcb.01342-09 ◽

2010 ◽

Vol 30 (7) ◽

pp. 1852-1863 ◽

Cited By ~ 18

Author(s):

Michael E. Engel ◽

Hong N. Nguyen ◽

Jolene Mariotti ◽

Aubrey Hunt ◽

Scott W. Hiebert

Keyword(s):

Gene Expression ◽

Notch Signaling ◽

Cell Fate ◽

Ex Vivo ◽

Notch Signaling Pathway ◽

Notch Receptor ◽

Intracellular Domain ◽

Fate Specification ◽

Dependent Cell ◽

Critical Components

ABSTRACT The Notch signaling pathway regulates gene expression programs to influence the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of the Notch receptor is cleaved by the γ-secretase complex and then translocates to the nucleus. There, it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional corepressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors, suggesting a pivotal role in regulation of the Notch transcription complex. The Notch1 intracellular domain disrupts the MTG16-CSL interaction. Ex vivo fate specification in response to Notch signal activation is impaired in Mtg16 −/− hematopoietic progenitors, and restored by MTG16 expression. An MTG16 derivative lacking the binding site for the intracellular domain of Notch1 fails to restore Notch-dependent cell fate. These data suggest that MTG16 interfaces with critical components of the Notch transcription complex to affect Notch-dependent lineage allocation in hematopoiesis.

Download Full-text

Bi-Directional Notch Signaling Via Signal-Sending and Signal-Receiving Microdomains of the Partnered Immunological Synapses during Th:DC Interaction.

Blood ◽

10.1182/blood.v108.11.646.646 ◽

2006 ◽

Vol 108 (11) ◽

pp. 646-646

Author(s):

Yatin M. Vyas ◽

Jerome Parness ◽

David Rodeberg ◽

Winifred Huang

Keyword(s):

Immune Responses ◽

Notch Signaling ◽

Information Transfer ◽

Notch Pathway ◽

Signal Propagation ◽

Cell Types ◽

Notch Receptor ◽

Cell Fate Decisions ◽

Notch Ligands ◽

Notch1 Receptor

Abstract The Notch pathway regulates adaptive immune responses, yet the temporal development of a specific molecular anatomy underlying the directionality of Notch signaling, central to cell-fate decisions, remains unknown. Using the development of the functional immune synapse (IS) of the human physiological T-helper lymphocyte (Th): Dendritic cell (DC) interaction as our model, we followed the temporal accumulation of Notch signaling components, unprocessed and processed, in the developing ThIS and the apposed DCIS of these cells by 2D and 3D immunofluorescense microscopy. Downstream Notch targets in both cell types were followed, as well. We demonstrate that Th-Notch1 receptor and DC-Notch ligands (Delta-like1, Jagged-1) cluster in their apposed central-supramolecular-activation-clusters (cSMAC), whereas DC-Notch1 receptor and Th-Notch ligands cluster in their apposed peripheral-SMAC in an anti-parallel arrangement to that seen in the cSMAC. The resultant accumulation in both cell types of processed nuclear Notch receptor, its ligands, as well as HES-1 and phosphorylated-STAT3, supports antiparallel, reciprocal Notch signal propagation in the DC-to-Th direction via the cSMAC and Th-to-DC direction via the pSMAC. The imposed asymmetric recruitment of the components of Notch pathway, therefore, provides a novel bi-directional route by which the partnered ThIS and DCIS regulate Notch-mediated immune responses. Our data indicate that terminally differentiated immune cells communicate bidirectionally using unidirectional Notch signaling platforms that are spatially segregated into reciprocally signaling microdomains. Significantly, our observations of bidirectional Notch signaling indicate that the heterologous Th:DC interaction is cooperative, requiring reciprocal information transfer across both cell types to mount an appropriate immune response.

Download Full-text

Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

10.1101/2022.01.13.476283 ◽

2022 ◽

Author(s):

LaNisha L. Patterson ◽

Thangam Sudha Velayutham ◽

Caitlan D. Byerly ◽

Duc Cuong Bui ◽

Jignesh Patel ◽

...

Keyword(s):

Notch Signaling ◽

Tandem Repeat ◽

Nuclear Translocation ◽

Peptide Mapping ◽

Notch Receptor ◽

Negative Regulator ◽

Receptor Interaction ◽

Linear Motif ◽

Notch Ligands ◽

Notch Activation

Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve exploitation of multiple evolutionarily conserved cellular signaling pathways including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/non-canonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands.

Download Full-text

Novel Notch signaling inhibitor NSI‑1 suppresses nuclear translocation of the Notch intracellular domain

International Journal of Molecular Medicine ◽

10.3892/ijmm.2019.4280 ◽

2019 ◽

Author(s):

Takaya Shiraishi ◽

Masahiro Sakaitani ◽

Satoko Otsuguro ◽

Katsumi Maenaka ◽

Toshiharu Suzuki ◽

...

Keyword(s):

Notch Signaling ◽

Nuclear Translocation ◽

Intracellular Domain ◽

Notch Intracellular Domain

Download Full-text

Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling

Development ◽

10.1242/dev.129.4.1049 ◽

2002 ◽

Vol 129 (4) ◽

pp. 1049-1059 ◽

Cited By ~ 6

Author(s):

Kenji Matsuno ◽

Mikiko Ito ◽

Kazuya Hori ◽

Fumiyasu Miyashita ◽

Satoshi Suzuki ◽

...

Keyword(s):

Notch Signaling ◽

Notch Pathway ◽

Notch Receptor ◽

Dominant Negative ◽

Mutant Form ◽

Dimerization Domain ◽

Intracellular Domain ◽

Signaling Mechanism ◽

Terminal Domain

The Notch pathway is an evolutionarily conserved signaling mechanism that is essential for cell-cell interactions. The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, we have analyzed the functional involvement of these Deltex domains in Notch signaling. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain was indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaved as a dominant-negative form. This dominant-negative Deltex inhibited Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting the dominant-negative Deltex might prevent the activation of the Notch receptor. We found that Deltex formed a homo-multimer, and mutations in the RING-H2 finger domain abolished this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupted the function of Deltex in vivo. However, when the same mutant was fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein had normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex.

Download Full-text