The Identity of the Notch Ankyrin Repeat Domain as a Determinant of Isoform-Specific Notch Signaling

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Project Summary/Abstract: The Notch signaling pathway is a crucial regulator of development and cell differentiation in almost all animal organs and tissues. The Notch receptor is a 300-kDa transmembrane protein consisting of an extracellular ligand-binding domain and an intracellular domain (NICD). The mammalian Notch pathways consists of four receptor isoforms which share a conserved architecture and overlapping but non-redundant functions. In fact, each isoform plays such a critical role in Notch function that congenital diseases due to severely deficient signaling by one isoform are exceedingly rare. The complexity encoded in the Notch pathway by the four receptor isoforms can be divided into two components: 1) isoform-specific differences in canonical Notch signaling outcomes, and 2) isoform-specific crosstalk with other signaling pathways. Upon receiving a signal, a series of proteolytic cleavages release the NICD from the membrane which, in the canonical pathway, translocates to the nucleus where it binds DNA-binding protein CSL and recruits co-activator Mastermind (MAML) to form the Notch transcriptional activation complex (NTC). Two domains of the NICD are responsible for forming the ternary NTC, the N-terminal disordered RAM linker and the ankyrin repeat domain (ANK) which forms a cleft with CSL for MAML binding. It has been suggested that isoform-specific outcomes in canonical signaling are mediated by variations in the ANK domains which are additionally responsible for mediating NTC dimerization on specialized promoters containing paired head-to-head CSL binding sites. However, these reports, to date, have been qualitative in nature and have offered inconsistent conclusions. Additionally, these studies did not investigate the role of the ANK domains in facilitating crosstalk with other pathways. Because ANK domains are ubiquitous protein-protein interaction motifs, it is unsurprising that many non-canonical NICD interactions are also mediated by the ANK domains. Two of the most common Notch-intersecting pathways are Wnt and TGFβ, and though this crosstalk is isoform-specific, the synergistic effects of Notch, Wnt, and TGFβ signaling have been implicated in numerous disease states, particularly in metastatic events and increased tumor invasion in breast cancer. Despite numerous attempts to therapeutically target Notch signaling, a lack of data on the mechanism governing isoform-specific Notch signaling has left these efforts unsuccessful. I propose three Aims to fill this void: I will 1) quantify the stability of each isoform’s NTC and their dimerization capacities, 2) characterize the binding of each ANK isoform with key non-canonical binding partners that crosstalk with Wnt and TGFβ, and 3) define the role of variations in the ANK domains on Wnt and TGFβ crosstalk in the cellular context of breast cancer. This work will integrate in vitro biophysical characterizations with functional analyses in cells to define the biophysical mechanism(s) and sequence basis of isoform-specific Notch signaling. These data represent a holistic view of Notch signaling that will serve as a critical foundation for designing more targeted, isoform-specific Notch therapeutics.