Hierarchical Phosphorylation within the Ankyrin Repeat Domain Defines a Phosphoregulatory Loop That Regulates Notch Transcriptional Activity

Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2011; 286(33):28844-57. DOI: 10.1074/jbc.M111.243600
Source: PubMed


The Notch signal transduction pathway mediates important cellular functions through direct cell-to-cell contact. Deregulation
of Notch activity can lead to an altered cell proliferation and has been linked to many human cancers. Casein kinase 2 (CK2),
a ubiquitous kinase, regulates several cellular processes by phosphorylating proteins involved in signal transduction, gene
expression, and protein synthesis. In this report we identify NotchICD as a novel target of phosphorylation by CK2. Using mapping and mutational studies, we identified serine 1901, located in
the ankyrin domain of Notch, as the target amino acid. Interestingly, phosphorylation of serine 1901 by CK2 appears to generate
a second phosphorylation site at threonine 1898. Furthermore, threonine 1898 phosphorylation only occurs when Notch forms
a complex with Mastermind and CSL. Phosphorylation of both threonine 1898 and serine 1901 resulted in decreased binding of
the Notch-Mastermind-CSL ternary complex to DNA and consequently lower transcriptional activity. These data indicate that
the phosphorylation of serine 1901 and threonine 1898 negatively regulates Notch function by dissociating the complex from
DNA. This study identifies a new component involved in regulation of NotchICD transcriptional activity, reinforcing the notion that a precise and tight regulation is required for this essential signaling

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    • "However, to date, mostly kinases that negatively regulate NIC1 function have been identified: AKT was shown to promote NIC1 cytoplasmic localization [49], phosphorylation of NIC1 by DYRK1A attenuated NOTCH signalling [50], and NLK-dependent NIC1 phosphorylation decreased the formation of the NIC1/CSL complex [51]. Capobianco group recently provided evidence that prior or during NIC1/CSL/MAML1 complex association, NIC1 is phosphorylated by CK2 allowing the accessibility to a subsequent phosphorylation site [52]. Both phosphorylation events appeared required for dissociation of the complex from DNA. "
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    ABSTRACT: Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "CyclinC:CDK8 recruitment to the complex promotes hyperphosphorylation of NotchICD within its PEST domain and targets the protein for Fbw7/Sel10 E3 ubiquitin ligase-dependent degradation (Fryer et al., 2004). Other kinases that promote NotchICD turnover through phosphorylation include the Wnt pathway associated enzyme glycogen synthase kinase 3β (GSK-3β; Espinosa et al., 2003) and the ubiquitously expressed casein kinase 2 (Ranganathan et al., 2011). Figure 1 provides an overview of the canonical Notch signaling pathway. "
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    ABSTRACT: Notch signaling in the cardiovascular system is important during embryonic development, vascular repair of injury, and vascular pathology in humans. The vascular smooth muscle cell (VSMC) expresses multiple Notch receptors throughout its life cycle, and responds to Notch ligands as a regulatory mechanism of differentiation, recruitment to growing vessels, and maturation. The goal of this review is to provide an overview of the current understanding of the molecular basis for Notch regulation of VSMC phenotype. Further, we will explore Notch interaction with other signaling pathways important in VSMC.
    Preview · Article · Apr 2012 · Frontiers in Physiology
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    ABSTRACT: Notch signaling plays a pivotal role in the regulation of many fundamental cellular processes, such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. At the molecular level, ligand binding induces the proteolytic cleavage of the Notch receptor. The intracellular domain of Notch translocates subsequently into the nucleus, associates with the central transcription factor RBP-J and activates transcription. Although, this pathway is remarkably short, with no second messenger involved, it regulates expression of more than hundred target genes in a tissue-specific manner. This review summarizes recent studies on transcriptional and chromatin control mechanisms, which set the stage for specific expression of Notch target genes. Furthermore, we review how the canonical (RBP-J dependent) Notch pathway is fine-tuned by downstream effectors and feedback loops in mammals.
    Full-text · Article · Jan 2012 · Cell cycle (Georgetown, Tex.)
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