N4BP1 mediates RAM domain-dependent notch signaling turnover during neocortical development

Notch signaling pathway activity, particularly fluctuations in the biologically active effector fragment NICD, is required for rapid and efficient dynamic regulation of proper fate decisions in stem cells. In this study, we identified NEDD4-binding protein 1 (N4BP1), which is highly expressed in the developing mouse cerebral cortex, as a negative modulator of Notch signaling dynamics in neural progenitor cells. Intriguingly, N4BP1 regulated NICD stability specifically after Notch1 S3 cleavage through ubiquitin-mediated degradation that depended on its RAM domain, not its PEST domain, as had been extensively and previously described. The CoCUN domain in N4BP1, particularly the Phe-Pro motif (862/863 amino acid), was indispensable for mediating NICD degradation. The Ring family E3 ligase Trim21 was, in contrast to other NEDD4 family members, required for N4BP1-regulated NICD degradation. Overexpression of N4BP1 in cortical neural progenitors promoted neural stem cell differentiation, whereas neural progenitor cells lacking N4BP1 were sensitized to Notch signaling, resulting in the maintenance of stem-like properties in neural progenitor cells and lower production of cortical neurons.

Automated summary

In this study, the negative modulator N4BP1 in neural progenitor cells was found to regulate the dynamics of Notch signaling pathway by mediating the stability of NICD after Notch1 S3 cleavage. The CoCUN domain in N4BP1, particularly the Phe-Pro motif, played a crucial role in mediating the degradation of NICD. Additionally, the E3 ligase Trim21 was specifically required for N4BP1-regulated NICD degradation. Overexpression of N4BP1 promoted neural stem cell differentiation, while the absence of N4BP1 sensitized neural progenitor cells to Notch signaling, resulting in the maintenance of stem-like properties and reduced production of cortical neurons in the cerebral cortex.