Abstract
Notch is a phylogenetically conserved transmembrane receptor that is required for many aspects of animal development. Upon ligand stimulation, a fragment of Notch is released proteolytically and enters the nucleus to form a complex with the DNA-binding protein CSL (CBF1/Suppressor of Hairless/Lag1) and activate transcription of Notch-CSL target genes. The physical structure of the Notch-CSL complex remains unclear, however, clouding the interpretation of previous efforts to correlate Notch structure and function. We have, therefore, characterized the binding of Drosophila CSL (called Suppressor of Hairless, or Su(H)) to the intracellular domain of Drosophila Notch both in vitro and in vivo. We report the identification of two Su(H) binding regions in Notch. The first is in the juxtamembrane region (the "RAM" domain). The second is just C-terminal to the Notch ankyrin repeats, overlapping or identical to two previously proposed nuclear localization sequences, in a domain we term PPD (potential phosphorylated domain). The ankyrin repeats themselves do not bind to Su(H); however, they substantially enhance binding of Su(H) to the more C-terminal region. Consistent with this picture, removal of either the Ram or PPD binding sites, separately, modestly reduces Notch activity in vivo, whereas removal of both renders Notch severely defective. These results clarify the relationship between Notch and CSL, help to explain the importance of the ankyrin repeats in Notch signaling, and reconcile many apparently contradictory results from previous Notch structure/function studies. Moreover, they suggest a second function for the Notch nuclear localization sequence elements.
Highlights
The intracellular domain of Drosophila Notch is large, 937 amino acids, and contains a variety of signaling domains and binding sites for regulators and effectors
Su(H) Interacts with Two Separate Regions of Nicd in Vitro—To map the Su(H) interaction region(s) within the Drosophila Notch intracellular domain (Nicd) in vitro, we purified GST-Su(H) fusion protein produced in SF9 cells infected by recombinant baculovirus, and we in vitro translated and 35Slabeled different functional regions of the Notch intracellular domain (Fig. 1A)
Whereas EGFP did not bind at all to GST-Su(H), EGFP-PPD bound GST-Su(H) about as well as did PPD-OPA (Fig. 1C, compare lanes 1 and 2). These data suggest that the PPD domain can interact with Su(H), but somehow this interaction is strongly improved by the presence of the ANK domain. These data suggest the existence of two separate Su(H) binding sites within Drosophila Nicd, one in the PPD domain that is sensitive to the presence of the ankyrin repeats and one in the RAM domain
Summary
Notch Fragments—His-tagged Notch fragments RAM and OPA were PCR-amplified and subcloned between the NheI and BglII sites of pRSET A and have been previously described [30]. PUAS NFL Wild Type—To get the pUAS Notch(full length) wild type we cleaned the stop mutation of pUAS N1–2262 [31] by replacing its XhoI/XbaI fragment by the one from pUAS, N1790 [16]. PUAS Su(H)-HA—The triple HA tag was derived from the vector pIRES-hrGFP2a (Stratagene), cut with XhoI and BstEII (blunt), and subcloned into pBS-SK-cut XhoI and KpnI (blunt). For all the mutations in the RAM domain, the NheI-NcoI fragment of pUAS N1–2262 was subcloned into pRSET and mutagenized. All the mutagenized fragments were fully sequenced before being re-subcloned in their appropriate backbones (pUAS Notch(full length) wild type, pUAS N1766 or N1779, pRSET N1766 or N1779, etc.)
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