NICD 152 still interacts with the RBPJ/MAML1 transactivation complex and the USP28/FBXW7 NICD degradation complex. (A) NICD variants demonstrate comparable binding capacities to RBPJ/MAML1 as revealed by co-IP. (B) Scheme of interaction between substrates and USP28/FBXW7. As a E3 ubiquitin ligase, FBXW7 targets substrates for proteasomal degradation by ubiquitination, whereas deubiquitinase USP28 is reported to antagonize FBXW7 leading to substrate stabilization.25 (C) Overview on protein sequences of C-terminal NICD variants. The CDC4 phospho-degron is shaded in blue, and the phospho-S (serine) sequence in orange. (D) Co-IP of Flag USP28 and NICD variants in NOTCH1 KO HEK 293 cells.24 Compared with NICD wt (lane 4), NICD 143 (lane 7) and NICD 152 (lane 13) but not NICD ΔCT (lane 10) showed stronger binding to USP28. (E) In NOTCH1 KO HEK 293 cells, NICD 143 (lane 3) and NICD 152 (lane 5) had higher affinity to endogenous USP28 compared with NICD wt (lane 2). (F) Sequences of GFP-tagged C-terminal NICD proteins (UniProt: P46531). The GFP tag is shown in green, common NICD sequence in orange, and amino acids which are different within the NICD variants in purple or dark blue. (G) The C terminus of NICD 152 (GFP Y2490 152, lane 3) still demonstrated higher affinity to USP28 in the co-IP performed in HEK 293 wt cells compared with C-terminal NICD wt (GFP Y2490 wt, lane 2) and NICD ΔCT (GFP Y2490 ΔCT, lane 4). (H) The C terminus of GFP Y2490 152 (lane 4) also demonstrated binding capacity to FBXW7 compared with GFP Y2490 wt (lane 3) but not GFP Y2490 ΔCT (lane 5) in the co-IP performed in HEK 293 wt cells. (I) Luciferase-based transcription assay. The pGA981-6 reporter (see also supplemental Figure 6), NICD wt (for preactivation), GFP Y2490 wt, GFP Y2490 152, and GFP Y2490 ΔCT were co-transfected in HeLa wt cells. Normalized luciferase activities (to NICD wt) revealed that the C terminus of NICD 152 (GFP Y2490 152) led to the rise of NICD wt transcription activities but not GFP Y2490 wt and GFP Y2490 ΔCT. Mean ± SD; n = 8; 2-tailed unpaired t test. − indicates the absence of corresponding proteins, + denotes the presence of respective proteins, and ∗ represents the heavy chains of antibodies. mRBPJ, murine RBPJ; ns, not significant.
Figure 5.

NICD 152 still interacts with the RBPJ/MAML1 transactivation complex and the USP28/FBXW7 NICD degradation complex. (A) NICD variants demonstrate comparable binding capacities to RBPJ/MAML1 as revealed by co-IP. (B) Scheme of interaction between substrates and USP28/FBXW7. As a E3 ubiquitin ligase, FBXW7 targets substrates for proteasomal degradation by ubiquitination, whereas deubiquitinase USP28 is reported to antagonize FBXW7 leading to substrate stabilization.25 (C) Overview on protein sequences of C-terminal NICD variants. The CDC4 phospho-degron is shaded in blue, and the phospho-S (serine) sequence in orange. (D) Co-IP of Flag USP28 and NICD variants in NOTCH1 KO HEK 293 cells.24 Compared with NICD wt (lane 4), NICD 143 (lane 7) and NICD 152 (lane 13) but not NICD ΔCT (lane 10) showed stronger binding to USP28. (E) In NOTCH1 KO HEK 293 cells, NICD 143 (lane 3) and NICD 152 (lane 5) had higher affinity to endogenous USP28 compared with NICD wt (lane 2). (F) Sequences of GFP-tagged C-terminal NICD proteins (UniProt: P46531). The GFP tag is shown in green, common NICD sequence in orange, and amino acids which are different within the NICD variants in purple or dark blue. (G) The C terminus of NICD 152 (GFP Y2490 152, lane 3) still demonstrated higher affinity to USP28 in the co-IP performed in HEK 293 wt cells compared with C-terminal NICD wt (GFP Y2490 wt, lane 2) and NICD ΔCT (GFP Y2490 ΔCT, lane 4). (H) The C terminus of GFP Y2490 152 (lane 4) also demonstrated binding capacity to FBXW7 compared with GFP Y2490 wt (lane 3) but not GFP Y2490 ΔCT (lane 5) in the co-IP performed in HEK 293 wt cells. (I) Luciferase-based transcription assay. The pGA981-6 reporter (see also supplemental Figure 6), NICD wt (for preactivation), GFP Y2490 wt, GFP Y2490 152, and GFP Y2490 ΔCT were co-transfected in HeLa wt cells. Normalized luciferase activities (to NICD wt) revealed that the C terminus of NICD 152 (GFP Y2490 152) led to the rise of NICD wt transcription activities but not GFP Y2490 wt and GFP Y2490 ΔCT. Mean ± SD; n = 8; 2-tailed unpaired t test. − indicates the absence of corresponding proteins, + denotes the presence of respective proteins, and ∗ represents the heavy chains of antibodies. mRBPJ, murine RBPJ; ns, not significant.

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