Among the defined domains of Notch, the ankyrin (CDC10) repeats of the intracellular portion, represent a potentially important site for protein-protein interactions. Drosophila Notch-derived ankyrin repeat containing peptides have been shown to bind the cytoplasmic protein, Deltex. The Deltex-Notch interaction prevents the observed cytoplasmic retention of the suppressor of hairless (Su [H]) protein by Notch ankyrin repeats, resulting in nuclear translocation. Mouse Notch ankyrin repeats associate with the human analogue of Su (H) / CBF-1 and act as transcriptional activators. The intracellular domain of Notch/TAN-1 contains six ankyrin repeats that have homology with the IkB family of proteins. A common structural feature of the IkB proteins is the presence of five to seven closely adjacent an ankyrin repeats. IkB proteins are specific inhibitors of NF-kB Rel transcription factors. These include IkB-a, IkB-beta, Bcl-3, and the NF-kB p100 and p105. Upon activation, IkB proteins undergo a phosphorylation-controlled proteolysis that allows the release NF-kB /Rel factor to enter the nucleus and effect transactivation. The structural similarity between the cytoplasmic portion of TAN-1 and IkB proteins prompted us to investigate the potential of TAN-1 to act as a membrane-bound and, ultimately, cytoplasmically-released form of an IkB-like regulator of NF-kB. A GST-fusion protein, corresponding to the Notch/TAN-1 intracellular domain containing six ankyrin repeats and N- and C-terminal flanking sequences of TAN-1 (AA 1774-2230) was produced. Construction of this GST-fusion protein permitted production of an antibody specific for the intracellular region of Notch and reagents for the characterization of in vitro interactions with specific intracellular components of T-cells. The recombinant intracellular portion of TAN-1 was found to: 1) Specifically inhibit the DNA-binding activity of p-50 containing NF-kB complexes, 2) Functionally modulate kB-dependent transactivation, and 3) physically associate with NF-kB containing p50 and p65 subunits in a T-cell line. These observations show that TAN-1 may directly signal NF-kB transcription factors and modulate nuclear gene expression. These findings represent the first biochemical evidence of notch function in vertebrates and suggest that the oncogenic effects of activated notch/TAN-1 may be mediated through interactions with NF-kB. They also represent the first documentation of a membrane protein functioning as an IkB-like regulatory molecule. Current efforts are focused on more detailed analysis of the interaction surface of notch/TAN-1 with NF-kB and subcellular localization of TAN-1/NF-kB. In addition, we are examining the potential for interaction with other rel/NF-kB proteins. The participation of Notch as a putative regulator of HIV expression via LTR-NF-kB interaction is being investigated.
