title: Evidence that C promoter-binding factor 1 binding is required for Notch-1-mediated repression of activator protein-1.
abstract: Cell fate determination in invertebrate and vertebrate systems is regulated by the Notch signaling pathway. Four mammalian Notch genes, Notch 1-4, encode differentially expressed transmembrane receptors. The canonical Notch pathway involves proteolytic liberation of the Notch-1 intracellular domain (NIC-1), which activates CSL (CBF1, Su(H), and Lag-1)-mediated transactivation. We showed previously that NIC-1 also represses activator protein-1 (AP-1)-mediated transactivation. The N-terminal RAM (RBP-Jkappa associated molecule) domain of NIC-1 was required for both activation and repression. To investigate the mechanism of AP-1 repression, we tested whether distinct sequences within the RAM domain mediate activation versus repression. We analyzed the capacity of RAM domain mutants to bind endogenous CBF1, to activate CSL-mediated transactivation, and to repress AP-1. A mutant lacking 20 amino acids of the RAM domain (Delta1759-1778) resembled the RAM domain deletion mutant in being defective in all activities. Analysis of 14 deletion and alanine substitution mutants revealed a correlation between CBF1 binding, CSL-mediated transactivation, and AP-1 repression. Stably transfected K562 cells could only tolerate very low level expression of wild-type NIC-1 and NIC-1 mutants retaining activation/repression activities. By contrast, transcriptionally compromised NIC-1 mutants accumulated at high levels. These results support a model in which the binding of NIC-1 to CBF1 is required for AP-1 repression and reveal a powerful cell-sensing mechanism that suppresses the levels of transcriptionally competent NIC-1.
authors.affiliation: Department of Pharmacology, Molecular and Cellular Pharmacology Program, University of Wisconsin Medical School, 383 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USA.
authors.forename: Jianlin
authors.initials: J
authors.lastname: Chu
authors.forename: Emery H
authors.initials: EH
authors.lastname: Bresnick
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D015153:Blotting, Western; D019070:Cell Lineage; D002462:Cell Membrane / Q000378:metabolism; D054407:Chemokine CCL4; D019742:Chemokines, CC; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D029721:Drosophila Proteins / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D020014:K562 Cells; D019402:Macrophage Inflammatory Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry
chemical_list: C066507:CCL4 protein, human; D054407:Chemokine CCL4; D019742:Chemokines, CC; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D019402:Macrophage Inflammatory Proteins; C497761:NOTCH1 protein, human; D009687:Nuclear Proteins; D011506:Proteins; C497570:RBPJ protein, human; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D012097:Repressor Proteins; C088731:Su(H) protein, Drosophila; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D000409:Alanine