Regulation of Notch1 signaling by Delta-like ligand 1 intracellular domain through physical interaction - PubMed (original) (raw)

Regulation of Notch1 signaling by Delta-like ligand 1 intracellular domain through physical interaction

Jane Jung et al. Mol Cells. 2011 Aug.

Abstract

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. The Delta-like ligand 1 also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. In this study, we have demonstrated that the Delta-like 1 intracellular domain (Dll1-IC) specifically binds to Notch1-IC in the nucleus, thereby disrupting the association of the Notch1-IC-RBP-Jk-MAM transcription activator complex. Additionally, the Notch1-mediated blockage of the induction of MyoD is abolished by the co-expression of Dll1-IC. Collectively, our results show that Dll1-IC functions as a negative regulator in Notch signaling via the disruption of the Notch1-IC-RBP-Jk complex.

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Figures

Fig. 1.. Dll1-IC suppresses the transcriptional activity of Notch1 target genes. (A, B, and C) HEK293 cells were transfected with expression vectors encoding for Notch1-IC and Dll1-IC with the luciferase reporter plasmids 4× CSL-Luc, Hes1-luc, and Hes5-luc along with pCMV-β-galactosidase as indicated. After 48 h of transfection, cells were lysed and assayed for luciferase activity. The luciferase activity of each sample was normalized to the β-galactosidase activity measured in the same sample. The data are expressed as the means ± S.D. of triplicates from one of three independent experiments. The data were evaluated for significant differences via student’s _t_-test. *ANOVA, P < 0.001.

Fig. 2.. Dll1-IC prevents the physical interaction between Notch1-IC and RBP-Jk. (A) HEK293cells were transfected with the indicated combinations of expression vectors for Myc-Notch1-IC, HA-RBP-Jk and GFPDll1-IC. The cell lysates were lysed and immunoprecipitated against anti-Myc monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-HA monoclonal antibody. The expression of Notch1-IC or RBPJk was analyzed via immunoblotting using anti-Myc or anti-HA monoclonal antibody. (B) HEK293 cells were transfected with the indicated combinations of expression vectors for Myc-Notch1-IC, Flag-RBP-Jk, HA-Mastermind and GFP-Dll1-IC. The cell lysates were then lysed and immunoprecipitated against anti-Myc monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Flag or anti-HA monoclonal antibody. Notch1-IC, RBP-Jk and Mastermind-like (MAML) expression were analyzed via immunoblotting using anti-Myc anti-Flag or anti-HA monoclonal antibodies.

Fig. 3.. Physical interaction of Dll1-IC with Notch1-IC in intact cells. (A) HEK293 cells were transfected with expression vectors encoding for Myc-Notch1-IC and GFP-Dll1-IC as indicated. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with anti-Myc antibody. The immunoprecipitates were then immunoblotted with anti-GFP antibody. Cell lysates were also immunoblotted with anti-Myc and anti-GFP antibody. (B) HEK293 cells were transfected with the expression vector encoding for Flag-Notch1-IC, Flag-Notch1-IC-N, Flag-Notch1-IC-NC, Flag-Notch1-IC-C, and GFP-Dll1-IC. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The immunoprecipitates were subsequently immunoblotted with anti-GFP antibody. Cell lysates were also immunoblotted with anti-Flag or anti-GFP antibody. (C) HEK293 cells were transfected with Flag-Notch1-IC, Myc-RBP-Jk or GFPDll1-IC. After 48 h, Flag-Notch1-IC and Myc-RBP-Jk were stained with Alexa-546. The cells were observed with a confocal microscope.

Fig. 4.. The effect of Dll1-IC on the suppression of the MyoD gene promoter activity by Notch1-IC. HEK293 cells were transfected with expression vectors encoding for Notch1-IC and Dll1-IC with the luciferase reporter plasmid MyoD-Luc along with pCMV-β-galactosidase as indicated. After 48 h of transfection, the cells were lysed and assayed for luciferase activity. The luciferase activity of each sample was normalized to the β-galactosidase activity measured in the same sample. These results represent the means ± S.D. of three independent experiments. The data were evaluated for significant differences by student’s _t_-test. *ANOVA, P < 0.05.

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Regulation of Notch1 signaling by Delta-like ligand 1 intracellular domain through physical interaction - PubMed (original) (raw)