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Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain

Nature volume 393, pages 382–386 (1998)Cite this article

Abstract

Notch proteins are ligand-activated transmembrane receptors involved in cell-fate selection throughout development1,2,3. No known enzymatic activity is contained within Notch and the molecular mechanism by which it transduces signals across the cell membrane is poorly understood. In many instances, Notch activation results in transcriptional changes in the nucleus through an association with members of the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su(H), Lag-1)1,2,3,4. As Notch is located in the plasma membrane and CSL is a nuclear protein, two models have been proposed to explain how they interact (Fig. 1) . The first suggests that the two interact transiently at the membrane1,5,6,7. The second postulates that Notch is cleaved by a protease, enabling the cleaved fragment to enter the nucleus6,8,9,10,11,12,13,14. Here we show that signalling by a constitutively active membrane-bound Notch-1 protein requires the proteolytic release of the Notch intracellular domain (NICD), which interacts preferentially with CSL. Very small amounts of NICD are active, explaining why it is hard to detect in the nucleus in vivo. We also show that it is ligand binding that induces release of NICD.

a, Models not invoking processing propose that interactions between Notch and CSL at the membrane may be sufficient to transduce a signal. Thus, ligand-regulated NICD release is not expected to be necessary for signalling. b, The processing model suggests that Notch signalling requires the release of NICD, which is capable of direct interaction with CSL in the nucleus, to turn on transcription of target genes. This model predicts that NICD release is regulated by ligand binding and that blocking proteolysis interferes with signalling. Notch-1 domain symbols as in Fig. 2.

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Figure 2: The Notch-1 constructs used and processing of NΔE to produce NICD.
Figure 3: Western blot analysis.
Figure 4: Activation of HES-1–luc by Notch-1 proteins.
Figure 5: Notch-1 acts below threshold of detection.
Figure 6: Full-length Notch-1 is cleaved when expressed together with Jagged-1.

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Acknowledgements

We thank A. Israel for providing us with the FLAG–CSLRBP3 expression vector; I.Frolov for the Sindbis virus vectors pSINrep5 and DHBB; G. Rubin and D. J. Pan for the Kuzbanian cDNAs; D. Syder and M. Crankshaw for technical help; R. Cagan, I. Boime, J. Gordon, D. Ornitz and members of the Kopan lab for reading and commenting on the manuscript; and G. Goldberg, G. Grant and B. Marmer for discussions and technical advice. R. K. Thanks H. Weintraub for encouragement and support. This research was supported by the NIH.

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  1. Division of Dermatology and the Department of Molecular Biology and Pharmacology, Washington University, Box 8123, 4940 Parkview Place, St Louis, 63110, Missouri, USA

    Eric H. Schroeter, Jeffrey A. Kisslinger & Raphael Kopan

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  1. Eric H. Schroeter
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Correspondence to Raphael Kopan.

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Schroeter, E., Kisslinger, J. & Kopan, R. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382–386 (1998). https://doi.org/10.1038/30756

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