Quantitative Dissection of the Notch:CSL Interaction: Insights into the Notch-mediated Transcriptional Switch

Olga Y. Lubman, Ma Xenia G. Ilagan, Raphael Kopan, Doug Barrick

Research output: Contribution to journalArticlepeer-review

64 Scopus citations

Abstract

Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the ∼ 140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (≤ 25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD.

Original languageEnglish
Pages (from-to)577-589
Number of pages13
JournalJournal of Molecular Biology
Volume365
Issue number3
DOIs
StatePublished - Jan 19 2007

Keywords

  • CSL
  • Notch intracellular domain
  • Notch signaling
  • isothermal titration calorimetry
  • transcriptional activation

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