title: Dissecting and circumventing the requirement for RAM in CSL-dependent Notch signaling.
abstract: The Notch signaling pathway is an intercellular communication network vital to metazoan development. Notch activation leads to the nuclear localization of the intracellular portion (NICD) of the Notch receptor. Once in the nucleus, NICD binds the transcription factor CSL through a bivalent interaction involving the high-affinity RAM region and the lower affinity ANK domain, converting CSL from a transcriptionally-repressed to an active state. This interaction is believed to directly displace co-repressor proteins from CSL and recruit co-activator proteins. Here we investigate the consequences of this bivalent organization in converting CSL from the repressed to active form. One proposed function of RAM is to promote the weak ANK:CSL interaction; thus, fusion of CSL-ANK should bypass this function of RAM. We find that a CSL-ANK fusion protein is transcriptionally active in reporter assays, but that the addition of RAM in trans further increases transcriptional activity, suggesting another role of RAM in activation. A single F235L point substitution, which disrupts co-repressor binding to CSL, renders the CSL-ANK fusion fully active and refractory to further stimulation by RAM in trans. These results suggest that in the context of a mammalian CSL-ANK fusion protein, the main role of RAM is to displace co-repressor proteins from CSL.
authors.affiliation: TC Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Johnson
authors.forename: Douglas
authors.initials: D
authors.lastname: Barrick
mesh_terms: D019943:Amino Acid Substitution; D017089:Ankyrin Repeat; D002460:Cell Line; D056970:Co-Repressor Proteins / Q000378:metabolism; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D062105:Molecular Docking Simulation; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D056970:Co-Repressor Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C085914:NUP214 protein, human; D028861:Nuclear Pore Complex Proteins; C497570:RBPJ protein, human; D016601:RNA-Binding Proteins; D051880:Receptors, Notch; D014157:Transcription Factors; C569288:RAMAC protein, human