Study of Intracellular Regulators of Notch Signaling

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We had identified a human homolog of Mastermind (Man), which had been implicated as an important positive regulator of Notch signaling pathway by genetic analyses in Drosophila (hMam-1). During this research project, we have further identified two new members of human Man family (hMam-2 and hMam-3), which retain characteristics similar to hMam-1 and Drosophila Mam. Both hMam-2 and hMam-3 stabilize and participate in the DNA-binding complex RBP-J protein and the Notch intracellular domains that serve as intermediates of the signaling. Both hMam-2 and hMam-3 enhanced the activation of transcription from a target promoter by Notch signaling. However, we also obtained evidence that the activation of the target promoter by Notch3 and Notch4 is more efficiently potentiated by hMam-2 than by hMam-1 or -3. Thus the multiplicity of Mam proteins in the mammalian system may help provide divergence to the strength of the Notch signals in different cell types.We have also investigated function of a novel maternal gene, neurotic, which is suggested to be an essential component for Notch signaling, neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats. We have found Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. Our results suggest that O-fucosylation catalyzed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions. This is an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction.