Related Experiment Videos
Murine notch homologs (N1-4) undergo presenilin-dependent proteolysis
M T Saxena1, E H Schroeter, J S Mumm
1Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110, USA.
Abstract:
Oncogenic forms of Notch1, Notch2, and Notch4 appear to mimic signaling intermediates of Notch1 and suggest that the role of proteolysis in Notch signaling has been conserved. Here we demonstrate that extracellularly truncated Notch homologs are substrates for a presenilin-dependent gamma-secretase activity. Despite minimal conservation within the transmembrane domain, the requirement for a specific amino acid (P1' valine) and its position at the cleavage site relative to the cytosolic border of the transmembrane domain are preserved. Cleaved, untethered Notch intracellular domains from each receptor translocate to the nucleus and interact with the transcriptional regulatory protein CSL. All four Notch proteins display presenilin-dependent transactivating potential on a minimal promoter reporter. Thus, this study increases the number of biochemically characterized gamma-secretase substrates from two to five. Despite a high degree of structural homology and the presenilin-dependent activity of truncated Notch proteins, the extent that this reflects functional redundancy is unknown.
Insights
Extracellularly truncated Notch proteins are substrates for presenilin-dependent gamma-secretase activity, similar to Notch1. This conserved proteolysis mechanism allows Notch intracellular domains to activate gene transcription, expanding known gamma-secretase substrates.
Area of Science:
- Molecular Biology
- Cell Signaling
- Proteolysis
Background:
- Oncogenic Notch receptors (Notch1, Notch2, Notch4) suggest conserved proteolysis in Notch signaling.
- Presenilin-dependent gamma-secretase activity is crucial for Notch signaling pathway.
- Previous studies identified limited gamma-secretase substrates.
Purpose of the Study:
- To investigate if extracellularly truncated Notch homologs are substrates for presenilin-dependent gamma-secretase.
- To identify conserved features of the cleavage site across different Notch homologs.
- To determine the functional consequence of Notch proteolysis on nuclear translocation and transcriptional activity.
Main Methods:
- Biochemical assays to test gamma-secretase activity on truncated Notch proteins.
- Analysis of amino acid sequence and position at the cleavage site.
- Nuclear translocation assays for cleaved Notch intracellular domains.
- Reporter gene assays to measure transactivating potential.
Main Results:
- Extracellularly truncated Notch1, Notch2, and Notch4 are substrates for presenilin-dependent gamma-secretase.
- A conserved P1' valine at the cleavage site is critical for gamma-secretase activity.
- Cleaved Notch intracellular domains translocate to the nucleus and bind to CSL.
- All four Notch proteins exhibit presenilin-dependent transactivating potential.
Conclusions:
- This study identifies three new gamma-secretase substrates, increasing the total to five.
- The findings highlight conserved mechanisms of Notch proteolysis and gamma-secretase activity.
- The functional redundancy among Notch proteins mediated by gamma-secretase activity remains to be fully elucidated.