The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity.
Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.
Beatus
P
P
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden.
Lundkvist
J
J
Oberg
C
C
Pedersen
K
K
Lendahl
U
U
D021581:Active Transport, Cell Nucleus; D000818:Animals; D017487:Ankyrins / Q000737:chemistry*; D001667:Binding, Competitive; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D015398:Signal Transduction; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017487:Ankyrins; D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; C106075:NOTCH4 protein, human; C497762:Notch1 protein, mouse; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497570:RBPJ protein, human; C497571:Rbpj protein, mouse; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D014157:Transcription Factors; C073572:Notch4 protein, mouse; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase