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Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex.

Ligand binding by Notch receptors triggers a series of proteolytic cleavages that liberate the intracellular portion of Notch (ICN) from the cell membrane, permitting it to translocate to the nucleus. Nuclear ICN binds to a highly conserved DNA-binding transcription factor called CSL (also known as RBP-Jkappa, CBF1, Suppressor of Hairless, and Lag-1) and recruits Mastermind-like transcriptional co-activators to form a transcriptional activation complex. Using bioinformatics tools, we identified a Rel homology region (RHR) within CSL that was used as a guide to determine the minimal protein requirements for ternary complex formation. The RHR of CSL contains both the N- and C-terminal beta-sheet domains (RHR-n and RHR-c) of typical Rel transcription factors, as judged by circular dichroism spectra. Binding of monomeric CSL to DNA requires the entire RHR of CSL and an additional 125-residue N-terminal sequence, whereas binding to ICN requires only the RHR-n domain. Although the RAM (RBP-Jkappa (recombination-signal-sequence-binding protein for Jkappa genes)-associated molecule) domain of ICN is flexible and relatively unstructured as an isolated polypeptide in solution, it associates stably with CSL on DNA. Recruitment of Mastermind-like 1 (MAML1) to CSL.ICN complexes on DNA requires inclusion of the ankyrin repeat domain of ICN, and N- and C-terminal sequences of CSL extending beyond the DNA-binding region. The requirement for cooperative assembly of the MAML1.ICN.CSL.DNA complex suggests that a primary function of ICN is to render CSL competent for MAML loading. On the basis of our results, we present a working structural model for the organization of the MAML1.ICN.CSL.DNA complex.

Pubmed ID: 12644465

Authors

  • Nam Y
  • Weng AP
  • Aster JC
  • Blacklow SC

Journal

The Journal of biological chemistry

Publication Data

June 6, 2003

Associated Grants

  • Agency: NCI NIH HHS, Id: CA82308
  • Agency: NCI NIH HHS, Id: CA92433
  • Agency: NHLBI NIH HHS, Id: HL61001

Mesh Terms

  • Ankyrins
  • Biophysical Phenomena
  • Biophysics
  • DNA
  • DNA-Binding Proteins
  • Humans
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein
  • Membrane Proteins
  • Molecular Sequence Data
  • Nuclear Proteins
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-rel
  • Receptor, Notch1
  • Receptors, Cell Surface
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Trans-Activators
  • Transcription Factors
  • Transcriptional Activation