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, 20 (18), 6913-22

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

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

K Shimizu et al. Mol Cell Biol.

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 time-dependent manner. Finally, binding of DSL proteins to Notch2 also activated the transcription of reporter genes driven by the RBP-Jkappa-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.

Figures

FIG. 1
FIG. 1
Binding of full-length DSL proteins to Notch2 on the BaF3 cell surface. (A) Generation of three kinds of DSL-CHO(r) cell lines expressing full-length Delta1, Jagged1, and Jagged2. Expression of each full-length DSL protein was verified by Western blot analysis. fD1, fD1-CHO; fJ1, fJ1-CHO; fJ2, fJ2-CHO. (B) Binding of three DSL-CHO(r) cells to BaF3 was examined in a cell-cell association assay. adhered, BaF3 which adhered to CHO cells; nonadhered, BaF3 which did not adhere to CHO cells. (C) Binding of three membrane-bound DSL proteins to Notch2 on the BaF3 cell surface was verified by the methods described for coprecipitation using membrane-bound DSL proteins in Materials and Methods in the absence (lanes 1 to 3) or presence (lanes 4 to 9) of the cross-linking reagent DSG. To identify the Notch2 protein fragments, these lysates were precipitated with an anti-Notch2 rabbit polyclonal antibody (lanes 7 to 9). To show the size difference between BaF3-Notch2TM and CHO-Notch2TM, lysates of these cells were separately precipitated with an anti-Notch2 rabbit polyclonal antibody (lanes 10 and 11). BaF3-Notch2TM, BaF3-derived Notch2TM; CHO-Notch2TM, CHO(r)-derived Notch2TM.
FIG. 2
FIG. 2
Preparation of soluble DSL proteins comprising a full-length extracellular region tagged with Fc or Flag(His)6. Three kinds of Fc-fused DSL proteins and two kinds of Flag(His)6-tagged DSL proteins produced in CHO(r) cells were purified with protein G or Ni beads, respectively. Integrity and purity were verified by Coomassie brilliant blue (CBB) staining in reducing and nonreducing conditions for sD1-Fc, sJ1-Fc, and sJ2-Fc (A) and by CBB staining and Western blot for sD1-Flag(His)6 and sJ1-Flag(His)6 (B).
FIG. 3
FIG. 3
Soluble Delta1, Jagged1, and Jagged2 protein binding to Notch2 present on the cell surface. (A) Three DSL-Fc proteins were allowed to bind to BaF3 at the same molar concentration (33 nM). As a control, the same concentration of hIgG was used. (B) Binding of increasing concentrations of three DSL proteins to BaF3. The extent of fluorescence brightness that gives the highest frequency (vertical axis) was plotted against the concentration of sD1-Fc, sJ1-Fc, and sJ2-Fc (horizontal axis). (C) Requirement of Ca2+ in binding of soluble DSL proteins to BaF3. BaF3 was incubated with each DSL protein in the absence (green) or presence (red) of 2 mM EGTA. As a control, hIgG was incubated with the cells in the absence of EGTA (black). (D) Self-displacement and reciprocal displacement of soluble Delta1 and soluble Jagged1. (a) Displacement of sJ1-Fc binding to BaF3 by a 500-fold molar excess of sD1-Flag(His)6 or sJ1-Flag(His)6. (b) Displacement of sD1-Fc binding to BaF3 by a 500-fold molar excess of sD1-Flag(His)6 or sJ1-Flag(His)6. (E) Coprecipitation analysis. DSL-Fc (lane 1) and hIgG (lane 5) in cell-binding buffer were allowed to bind to BaF3. Protein G beads were then added directly to the BaF3 lysate to precipitate DSL-Fc-containing complex (lanes 1 to 4). To identify Notch2 protein fragments, the BaF3 lysate was precipitated with an anti-Notch2 rabbit polyclonal antibody (lanes 5). These precipitates were analyzed by a Western blot probing with a Notch2-specific monoclonal antibody. Size marker protein positions are shown on the left. Bands of approximate sizes of 120 kDa represent the membrane-spanning subunit (Notch2TM). fN2, full-length Notch2; Protein G, precipitation with Protein G alone.
FIG. 4
FIG. 4
Transduction of a Notch signal in BaF3 by full-length DSL proteins. BaF3 cells at 2 × 105 were transiently transfected with the PGa981-6 plasmid and were spread over a monolayer of CHO(r) cells stably expressing or not expressing various exogenous full-length DSL proteins. fD1, fD1-CHO; fJ1, fJ1-CHO; fJ2, fJ2-CHO.
FIG. 5
FIG. 5
Induction of cleavage, hyperphosphorylation, and nuclear accumulation of the intracellular domain of Notch2 by DSL proteins. (A) Cleavage of the membrane-spanning subunit of Notch2 and nuclear accumulation of intracellular domain of Notch2 by stimulation with various full-length DSL proteins. BaF3 cells were collected 1.5 h after coculture with CHO(r) lines stably expressing or not expressing various exogenous full-length DSL proteins. MC and N fractions were prepared as described in Materials and Methods, and in each fraction Notch2 fragments containing an intracellular domain were analyzed by Western blot using an anti-Notch2 antibody after immunoprecipitation. As controls for correct fractionation of the MC and N fractions, antibodies against IRβ for membrane proteins, insulin receptor substrate-2 (IRS-2) for cytosolic proteins, and Jun-D for nuclear proteins were used for each fraction in Western blot analysis. (B) Time-course analysis of Notch2 after stimulation with fD1-CHO. (C) Effect on Notch2 fragments of alkaline phosphatase (AP) treatment. From each MC and N fraction prepared from BaF3 after coculture with wild-type CHO(r) and fD1-CHO, Notch2 fragments containing the intracellular domain were immunoprecipitated. The samples were then treated by alkaline phosphatase and subjected to Western blotting.
FIG. 6
FIG. 6
Activation of the transcription by DSL proteins through exogenous Notch2. (A) Generation of CHO(r) expressing full-length Notch2 (fN2-CHO). Exogenous Notch2 in the fN2-CHO cells was verified by Western blot analyses using anti-Flag and anti-Notch2 monoclonal antibodies. fNotch2, unprocessed full-length Notch2; N2TM, membrane-spanning subunit of Notch2; EndoN2TM, endogenous Notch2TM fragment derived from CHO(r). (B and C) Reporter gene transactivation in CHO(r) with or without exogenous Notch2. CHO(r) cells with or without exogenous Notch2 were inoculated in a 24-well plate and transfected with three reporter plasmids: PGa981-6 (B), pHES1-luc, and pHES5-luc (C). The stimulators, CHO(r) lines stably expressing or not expressing various exogenous full-length DSL proteins, were then cocultured. In both panels B and C, the fold induction of luciferase activity in DSL-CHO(r) (mean of triplicate measurements with standard deviation) was calculated against that found in parental CHO(r) lines used as stimulators.

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