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      Notch and PKC Are Involved in Formation of the Lateral Region of the Dorso-Ventral Axis in Drosophila Embryos

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          Abstract

          The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

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          Toll-like receptors.

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            Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover.

            Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.
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              Nuclear access and action of notch in vivo.

              The Drosophila Notch (N) gene encodes a conserved single-pass transmembrane receptor that transduces extracellular signals controlling cell fate. Here, we present evidence that the intracellular domain of Notch gains access to the nucleus in response to ligand, possibly through a mechanism involving proteolytic cleavage and release from the remainder of the protein. In addition, our results suggest that signal transduction by Notch depends on the ability of the intracellular domain, particularly the portion containing the CDC10 repeats, to reach the nucleus and to participate in the transcriptional activation of downstream target genes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                4 July 2013
                : 8
                : 7
                : e67789
                Affiliations
                [1]Departments of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
                University of Otago, New Zealand
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CSW DMT. Performed the experiments: DMT CSW CJL SR. Analyzed the data: CSW DMT. Contributed reagents/materials/analysis tools: CSW DMT. Wrote the paper: CSW DMT.

                Article
                PONE-D-12-36764
                10.1371/journal.pone.0067789
                3701627
                23861806
                e416ed8c-6449-42f9-86ea-aab66ff8a68b
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 November 2012
                : 23 May 2013
                Page count
                Pages: 16
                Funding
                The study was supported by NIH 1 R21 HD062928 (to CSW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Computational Biology
                Signaling Networks
                Developmental Biology
                Molecular Development
                Signaling
                Model Organisms
                Animal Models
                Drosophila Melanogaster
                Molecular Cell Biology
                Signal Transduction
                Signaling in Selected Disciplines
                Developmental Signaling

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                Uncategorized

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