Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 23, Pages 21232-21239Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M301567200
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Funding
- NCI NIH HHS [CA92433, CA82308] Funding Source: Medline
- NHLBI NIH HHS [HL61001] Funding Source: Medline
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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.
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