Novel Biochemical Regulatory Mechanisms of Developmental Signaling Pathways

Abstract

The Notch signaling pathway is an essential cell-cell signaling pathway that is involved in cell fate decisions and cell differentiation during early metazoan development. In humans, the Notch pathway is misregulated in cancers such as T-cell acute lymphoblastic leukemia, breast cancer, lung cancer, and colorectal cancer. The key downstream effector of the Notch signaling pathway is the Notch Intracellular Domain (NICD), which is produced from a series of ligand-dependent proteolytic cleavages. Upon its liberation from the plasma membrane, NICD translocates into the nucleus and binds to the transcription factor CSL to activate a Notch-specific transcriptional program. Previous cultured cell studies suggest that regulated NICD turnover plays a critical role in regulating its steady-state intracellular levels and subsequent Notch pathway activation. Correspondingly, stabilizing mutations in NICD1 have been linked to multiple types of cancer. These results support the idea that regulation of NICD protein turnover is an essential process in regulation of Notch signaling. However, the mechanisms that control NICD degradation, and their effect on Notch signal transduction is a major unanswered question in the Notch field. We found that human NICD1 degrades robustly in a proteasome-dependent manner in Xenopus egg extract, and have identified a 35-amino acid degron (N-Box) at the N-terminus of NICD1. The N-Box is both sufficient and necessary for degradation of NICD1 in Xenopus egg extract, and, when attached to a heterologous protein in cultured human cells, promotes its turnover. Mutations in key residues within N-Box stabilize the NICD1 protein and lead to increased Notch transcriptional activity in cultured mammalian cells. We present a model as to how the N-Box may regulate NICD1 stability and transcriptional activity in the context of known stabilizing mutations of NICD1 in human cancers.