Binding of Delta1, Jagged1, and Jagged2 to Notch2 Rapidly Induces Cleavage, Nuclear Translocation, and Hyperphosphorylation of Notch2

Abstract

Delta1, Jagged1, and Jagged2, commonly designated Delta/Serrate/LAG-2 (DSL) proteins, are known to be ligands for Notch1. However, it has been less understood whether they are ligands for Notch receptors other than Notch1. Meanwhile, ligand-induced cleavage and nuclear translocation of the Notch protein are considered to be fundamental for Notch signaling, yet direct observation of the behavior of the Notch molecule after ligand binding, including cleavage and nuclear translocation, has been lacking. In this report, we investigated these issues for Notch2. All of the three DSL proteins bound to endogenous Notch2 on the surface of BaF3 cells, although characteristics of Jagged2 for binding to Notch2 apparently differed from that of Delta1 and Jagged1. After binding, the three DSL proteins induced cleavage of the membrane-spanning subunit of Notch2 (Notch2 TM ), which occurred within 15 min. In a simultaneous time course, the cleaved fragment of Notch2 TM was translocated into the nucleus. Interestingly, the cleaved Notch2 fragment was hyperphosphorylated also in a timedependent manner. Finally, binding of DSL proteins to Notch2 also activated the transcription of reporter genes driven by the RBP-J-responsive promoter. Together, these data indicate that all of these DSL proteins function as ligands for Notch2. Moreover, the findings of rapid cleavage, nuclear translocation, and phosphorylation of Notch2 after ligand binding facilitate the understanding of the Notch signaling.

Protein O -fucosyltransferase 1 is an essential component of Notch signaling pathways

“…Notch1 has Ͼ20 EGF repeats that contain a consensus sequence for O-fucosylation and most may be modified by Fringe (7). It is known that the action of Fringe in transferring GlcNAc to EGF-O-fucose causes changes in soluble ligand binding to Notch receptors (5,(13)(14)(15). We have shown that Lec13 mutant cells with low fucose in glycoproteins exhibit reduced Notch signaling for both Jagged1 and Delta1 ligands (refs.…”

“…It is found on urokinase-type plasminogen activator (uPA) (8), on Notch receptors and their ligands (6,9), on Cripto (10,11), and on other proteins, including blood clotting factors. O-Fucose is required for signaling by uPA through the uPA receptor (12), for Fringe modulation of Notch receptor signaling in Drosophila (4,5) and in coculture assays (4,(13)(14)(15)(16), and for Cripto to mediate Nodal signaling (10,11).…”

Murine Notch Homologs (N1–4) Undergo Presenilin-dependent Proteolysis

“…Post-translational phosphorylation of all Notch proteins was also observed (Fig. 3), N2 and N3 modifications have been previously noted (32,33). Although the Notch ⌬E constructs used in this paper include the analogous S2 cleavage site (Fig.…”

The C-terminal PDZ-Ligand of JAGGED1 Is Essential for Cellular Transformation

“…Where the specificity in DSL-Notch signaling lies is not well understood. Although it is not known which DSL proteins can activate which Notch molecules, the DSL proteins Jagged1, Jagged2, and Delta1 have been reported to bind Notch2 and subsequently induce processing, indicating that multiple DSL proteins can activate a specific Notch molecule (22). Furthermore, soluble forms of the extracellular domain of Jagged1 can physically interact with Notch1, Notch2, and Notch3 in binding assays (23,24).…”