TY - JOUR
T1 - Structural requirements for assembly of the CSL·intracellular Notch1·Mastermind-like 1 transcriptional activation complex
AU - Nam, Yunsun
AU - Weng, Andrew P.
AU - Aster, Jon C.
AU - Blacklow, Stephen C.
PY - 2003/6/6
Y1 - 2003/6/6
N2 - 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-Jκ, 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 β-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-Jκ (recombination-signal-sequence-binding protein for Jκ 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.
AB - 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-Jκ, 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 β-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-Jκ (recombination-signal-sequence-binding protein for Jκ 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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U2 - 10.1074/jbc.M301567200
DO - 10.1074/jbc.M301567200
M3 - Article
C2 - 12644465
AN - SCOPUS:0038079817
SN - 0021-9258
VL - 278
SP - 21232
EP - 21239
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 23
ER -