The life and death of the notch intracellular domain

Abstract

The Notch signaling pathway is an evolutionarily conserved way of communicating between cells that contact each other. Notch is a single-transmembrane spanning receptor that interacts with single-transmembrane spanning ligands on adjacent cells. In vertebrates the Notch family of receptor consists of four members: Notch1 to Notch4. The newly synthesized Notch receptor matures in the Golgi network and is transported to the plasma membrane. Upon interaction with ligand it is cleaved in two consecutive steps, the last of which is mediated by the gamma-secretase complex and results in release of the intracellular domain (NICD). The NICD translocates to the nucleus and interacts with the DNA-binding protein CSL resulting in increased transcription of Hes and Hey genes. After cleavage, Notch signaling can no longer be affected by extracellular cues and subsequent down-regulation must be at the level of the NICD. The work presented here focuses on three different ways to regulate NICD activity.

The first section deals with the divergence in the Notch receptor family, with particular focus on Notch1 and Notch3 ICDs. Notch3 ICD contains the same functional domains as Notch1 ICD but is a poor activator of transcription and can reduce Notch1 ICD-mediated transactivation. This could be a way of fine-tuning the activity of Notch ICD after cleavage, perhaps through competition for co-factors.

The second section focuses on protein degradation as a means of reducing NICD activity. Analysis of the mammalian E3 ubiquitin ligase Sel-10 showed that it participates in the ubiquitination and breakdown of NICD and reducing NICD activity following cleavage. Ubiquitination of NICD by Sel-10 is dependent on the most C-terminal PEST-containing region of Notch1 ICD. Sel-10 itself is subjected to ubiquitin-mediated degradation.

The third section of this thesis describes the cross-talk between Notch and the TGF- beta/BMP signaling pathway and how this interplay affects NICD activity. Smad3, a downstream mediator of TGF-beta signaling, acts synergistically with NICD to activate Notch target genes by binding to the NICD. Notch is an inhibitor of myogenesis and this is also the case for some ligands in the TGF-beta superfamily such as BMP. We found that the BMP-mediated block of myogenic differentiation is dependent on functional Notch signaling and that the BMP intracellular mediator Smad1 binds GC-rich stretches in the Hey1 promoter. Moreover, Smad1 interacts with NICD and CSL to increase Hey 1 reporter gene transcription. These are among the first examples of direct cross-talk between Notch and other major signaling pathways.