Mutational and energetic studies of Notch 1 transcription complexes

J Mol Biol. 2008 Feb 8;376(1):131-40. doi: 10.1016/j.jmb.2007.11.061. Epub 2007 Nov 28.

Abstract

Notch proteins constitute the receptors of a highly conserved signaling pathway that influences cell fate decisions both during development and in adulthood. A proteolytic cascade induced by ligand stimulation results in release of the intracellular Notch domain from the cell membrane, allowing it to enter the nucleus and form a complex with a DNA-bound transcription factor called CSL (CBF-1/RBP-J kappa, Suppressor of Hairless, and Lag-1) and a coactivator of the Mastermind family. Assembly of this Notch nuclear complex is the key step in the transcriptional response to a Notch signal. In the studies reported here, we mapped residues important for the stabilization of this multiprotein-DNA complex using site-directed mutagenesis, determined the affinity of the three-domain form of CSL for its various partners, and investigated sources of cooperativity in complex formation by monitoring the influence of various components of the complex on the interactions of CSL with its other partners. Our findings are consistent with a model for complex assembly in which the RBP-J kappa-associated molecule domain of Notch increases the effective concentration of the ankyrin domain for its binding site on the Rel-homology region of CSL, enabling docking of the ankyrin domain and subsequent recruitment of the Mastermind-like coactivator.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cell Line
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein / metabolism*
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / metabolism*
  • Protein Binding
  • Receptor, Notch1 / chemistry
  • Receptor, Notch1 / genetics
  • Receptor, Notch1 / metabolism*
  • Trans-Activators
  • Transcription Factors

Substances

  • DNA-Binding Proteins
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein
  • MAML1 protein, human
  • NOTCH1 protein, human
  • Nuclear Proteins
  • RBPJ protein, human
  • Receptor, Notch1
  • Trans-Activators
  • Transcription Factors
  • DNA