Abstract
Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome.
Highlights
The NOTCH receptors orchestrate a number of developmental processes besides ensuring adult tissue homeostasis [1,2]
Owing to the nature of the NOTCH receptors which are composed of two subunits held together non-covalently by calcium-dependent interactions [1,2], it was previously demonstrated that calcium depletion dissociates and activates the NOTCH receptors [28]
To begin delineating the mechanisms that account for the positive impact of the MEK/ERK pathway on NOTCH signalling, we investigated whether activation of the MEK/ERK pathway could promote the association of NIC1 with either CSL or MASTERMIND-LIKE 1 (MAML1)
Summary
The NOTCH receptors orchestrate a number of developmental processes besides ensuring adult tissue homeostasis [1,2]. This highly conserved signalling pathway has a relatively simple molecular architecture. The transmembrane NOTCH receptors (NOTCH 1-4) undergo sequential cleavages by ADAM-metalloproteases and the gamma-secretase complex. The latter, blocked by gammasecretase inhibitors, releases the NOTCH intracellular domain (NIC) that is free to translocate towards the nucleus to collaborate with the DNA-binding protein [CBF1, Su(H) and LAG-1] (CSL) and the co-activator MASTERMIND-LIKE 1 (MAML1) to modulate gene expression. One distinct characteristic of the NOTCH signalling pathway is the dual role of the receptor i.e. sensing the signal and achieving the response
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