Identification of a Paralog-Specific Notch1 Intracellular Domain Degron.

Matthew R Broadus,James G Patton,Tony W Chen,Jeanne N Jodoin,Leif R Neitzel,Stacey S Huppert,Victoria H Ng,Anthony J Capobianco,David J Robbins,Laura A Lee,Adrian Salic,Ethan Lee

doi:10.1016/j.celrep.2016.04.070

Abstract

Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism bywhich degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.

Highlights

The Notch pathway is a highly conserved, metazoan signaling pathway critical for organismal development (Kopan and Ilagan, 2009)
NICD1 Is Degraded in Xenopus Extract To recapitulate cytoplasmic Notch intracellular domain (NICD) turnover, we used the Xenopus extract system previously shown to support b-catenin degradation via Wnt pathway components (Chen et al, 2014)
We found that radiolabeled in-vitro-translated (IVT) hNICD1 degraded robustly when added to Xenopus extract (Figure 1A)

Summary

Introduction

The Notch pathway is a highly conserved, metazoan signaling pathway critical for organismal development (Kopan and Ilagan, 2009). The Notch pathway communicates transcriptional decisions between adjacent cells through direct interaction of a Delta/Serrate/Lag-2 (DSL) type 1 transmembrane ligand on the signaling cell and a Notch type 1 transmembrane receptor on a receiving cell. This interaction promotes a series of proteolytic events resulting in liberation of the Notch intracellular domain (NICD) from its membrane tether. Liberated NICD enters the nucleus, where it forms a complex with CSL (CBF1/RBPjk/Su(H)/ Lag-1), MAML (Mastermind-like), and CoA (coactivators) (Kovall and Blacklow, 2010). Formation of this complex drives transcription of Notch target genes. We identify a hNICD1-specific degron within the N-terminal region distinct from its C-terminal PEST domain

Methods

Results

Discussion

Conclusion