Research ArticleBiochemistry

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

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Science Signaling  05 Nov 2019:
Vol. 12, Issue 606, eaay2369
DOI: 10.1126/scisignal.aay2369

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How NRARP inhibits Notch signaling

The Notch-regulated ankyrin repeat protein (NRARP) is a feedback inhibitor of Notch signaling. Jarrett et al. found that NRARP inhibited the growth of Notch-dependent, T cell acute lymphoblastic leukemia cells and interacted with two members of the Notch transcriptional activation complex: the Notch intracellular domain (NICD) and the transcription factor RBPJ. The crystal structure of an NRARP-NICD-RBPJ-DNA complex showed that NRARP bound to both NICD and RBPJ through its ankyrin domains. Overexpression of NRARP in cells reduced the abundance of NICD, suggesting that NRARP may promote its degradation. These findings suggest how NRARP inhibits Notch-dependent transcriptional activation and identify the NRARP-NICD-RBPJ interaction as a potentially targetable node to inhibit Notch signaling in disease.

Abstract

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

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