Epidermal Growth Factor Receptor neddylation is regulated by a desmosomal-COP9 (Constitutive Photomorphogenesis 9) signalosome complex
- University of Detroit Mercy, United States
- Northwestern University, United States
Figures
Figure 1 with 4 supplements
The third subunit of the COP9 signalosome (CSN), Cops3, interacts with desmosomal components Desmoglein1 (Dsg1) and Desmoplakin (Dp).
(a) Cytotrap yeast two hybrid (Y2H) indicating an interaction between Cops3 and the cytoplasmic tail of Dsg1. Growth at 25°C is permissive; growth on galactose (Gal) at 37°C confirms interaction. (b)…
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Figure 1—source data 1
PLA analysis of Dsg1 and Cops3 in NHEKs differentiated for 1 day.
Quantification of raw data of PLA signal per cell for NHEKs cultures with siRNA-targeting COPS3, DSG1, DP, or scramble sequence (siCONT) after 1 day of differentiation. Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). *p<0.001 (Student’s t test); mean ±SEM (Figure 1g).
- https://doi.org/10.7554/eLife.22599.008
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Figure 1—source data 2
PLA analysis of Dp and Cops3 in NHEKs differentiated for 1 day.
Quantification of raw data of PLA signal per cell for NHEKs cultures with siRNA-targeting COPS3, DSG1, DP, or scramble sequence (siCONT) after 1 day of differentiation. Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). *p<0.00005 (Student’s t test); mean ±SEM (Figure 1h).
- https://doi.org/10.7554/eLife.22599.009
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Figure 1—source data 3
PLA analysis of desmosomal and adherens junctions components in NHEKs differentiated for 1 day.
Quantification of raw data of PLA signal per cell for NHEKs cultures with siRNA-targeting Cops3 (Figure 1—figure supplement 4a and b). Figure 1—figure supplement 4c displays the percent of PLA signal at cell junctions facilitated by co-staining of cell-cell junctions with Pg. Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis).
- https://doi.org/10.7554/eLife.22599.010
Figure 1—figure supplement 1
Cytotrap Y2H indicating no interaction between Cops3 and the N-terminal portion of Dp (amino acids 1–584).
Growth at 25°C is permissive; growth on galactose (Gal) at 37°C confirms interaction, while lack of growth on glucose (Glu) plates at 37°C excludes the presence of any false positives (ie. …
Figure 1—figure supplement 2
Western blots indicating knockdown efficiency of NHEKs used in proximity ligation assay (PLA) of Dsg1 and Cops3.
https://doi.org/10.7554/eLife.22599.005
Figure 1—figure supplement 3
Western blots indicating knockdown efficiency of NHEKs used in proximity ligation assay (PLA) of Dp and Cops3.
https://doi.org/10.7554/eLife.22599.006
Figure 1—figure supplement 4
Quantification of PLA analysis in NHEKS after 1 day of differentiation.
Antibody pairs are (a) Dsg1/Cops3, Dp/Cops3 and E-cad/B-cat, and Ecad/Cops3. *p<0.000005, Student’s t test; mean ±SEM. (b) Antibody pairs are adherens junction components Alpha Catenin (A-cat) and …
Figure 2 with 1 supplement
Keratinocyte differentiation requires Cops3.
(a) RNAi-mediated silencing of COPS3 in NHEK cultures differentiated for 2 days in 1.2mM Ca++results in a decrease in differentiation markers: Dsg1, Desmocollin1 (Dsc1), Keratin 10 (K10) and …
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Figure 2—source data 1
Cells transiently transfected with Dsg1-GFP do not express K10 upon the silencing of COPS3.
Quantification of raw data of average fluorescence intensity in Dsg1-GFP expressing cells. Quantification performed in FIJI (see sample size and statistical analysis). *p<0.00005 (Student’s t test); mean ±SEM (Figure 2g).
- https://doi.org/10.7554/eLife.22599.013
Figure 2—figure supplement 1
Multiple siRNA targets of COPS3 displayed keratinocyte differentiation defect and an increase in EGFR signaling in keratinocytes differentiated for 1 day in 1.2 mM Ca++.
https://doi.org/10.7554/eLife.22599.012
Figure 3
Silencing of Cops3 promotes Epidermal Growth Factor Receptor (EGFR) signaling in NHEKs.
(a) RNAi-mediated silencing of COPS3 in NHEKs differentiated for 2 days in 1.2 mM Ca++ with Human Keratinocyte Growth Factors (HKGF) display a decrease in differentiation markers (right panel) and …
Figure 4 with 2 supplements
Loss of desmosomal components, Cops3 or Nedd8 affects the biochemical status of EGFR.
(a) PLA analysis using Nedd8 and EGFR antibodies in NHEKs treated with siRNA targeting DP, DSG1, COPS3 or scramble (siCONT) (scale bar = 20 µm). Quantification of PLA signals displayed to the right …
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Figure 4—source data 1
PLA analysis of Nedd8/EGFR antibodies and Ubiqutin/EGFR antibodies in NHEKs.
Quantification of raw data of PLA signal per cell treated with siRNA targeting DP, DSG1, COPS3 or scramble (siCONT). Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). Nedd8/EGFR: *p<0.05 (Student’s t test); mean ±SEM (Figure 4a). Ubiquitin/EGFR: *p<0.05 (Student’s t test); mean ±SEM (Figure 4d).
- https://doi.org/10.7554/eLife.22599.018
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Figure 4—source data 2
Analysis of Nedd8-EGFR and Ubiquitin-EGFR through EGFR immunoprecipitations (IPs) in SCC9 cells.
Densitometry quantification of raw data of 4 biological replicates. Nedd8 and Ubiquitin quantifications were normalized to the total amount of EGFR immunoprecipitated from each biological replicate. Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). Nedd8-EGFR: *p<0.05 (Student’s t test); mean ±SEM (Figure 4b). Ubiquitin-EGFR: *p<0.01 (Student’s t test); mean ±SEM (Figure 4c).
- https://doi.org/10.7554/eLife.22599.019
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Figure 4—source data 3
PLA analysis of EGFR/Nedd8 and EGFR/Ub upon the loss of Nedd8.
Quantification of raw data of PLA signal per cell for NHEKs cultures with siRNA-targeting NEDD8 and COPS3 after 1 day of differentiation. Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). *p<0.00005 (Student’s t test); mean ±SEM (Figure 4—figure supplement 2).
- https://doi.org/10.7554/eLife.22599.020
Figure 4—figure supplement 1
Western blots indicating knockdown efficiency of NHEKs used in proximity ligation assay (PLA) of EGFR/Nedd8 and EGFR/Ub (Fig.
https://doi.org/10.7554/eLife.22599.016
Figure 4—figure supplement 2
EGFR is ubiquitinated upon loss of Nedd8 by PLA analysis in NHEKs differentiated for 1 day in high calcium.
(a) PLA analysis of NHEKs differentiated after 1 day with siRNA targeting NEDD8 and COPS3. Antibody pairings: AB-12 anti-EGFRMOUSE/Ab81264 anti-Nedd8RABBIT(Top panel) and FK1 anti-UbMOUSE/D38B1 …
Figure 5 with 1 supplement
Stability of EGFR and the receptor’s internalization are affected upon the loss of desmosomal components, Cops3 or Nedd8.
(a) NHEKs were treated in serum free media (1.2 mM Ca++), then treated with EGF (50 ng/ml) at indicated time points to assess the amount of internalized EGFR (scale bars = 20 μm). Quantification of …
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Figure 5—source data 1
EGFR localization upon stimulation in cells treated with siRNA targeting DP, DSG1, COPS3, or scramble (siCONT).
Quantification of raw data of EGFR fluorescence border intensity from 3 biological replicates. Quantification performed in FIJI (see sample size and statistical analysis). *p<0.01 (Student’s t test); mean ±SEM (Figure 5a).
- https://doi.org/10.7554/eLife.22599.023
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Figure 5—source data 2
EGFR localization upon stimulation in cells treated with siRNA targeting NEDD8, COPS3, or scramble (siCONT).
Quantification of raw data of EGFR fluorescence border intensity from 3 biological replicates. Quantification performed in FIJI (see sample size and statistical analysis). *p<0.01 (Student’s t test); mean ±SEM (Figure 5b).
- https://doi.org/10.7554/eLife.22599.024
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Figure 5—source data 3
Cycloheximide (CHX) treatment of NHEKS upon the loss of Nedd8.
Densitometry quantification of raw data of 3 biological replicates. Total EGFR protein expression was normalized to loading control GAPDH for each indicated time point. Quantification performed in FIJI (see sample size and statistical analysis). 8 hr CHX treatment = *p<0.02; and 24 hr CHX treatment = *p<0.01. Student’s t test; mean ±SEM (Figure 5c).
- https://doi.org/10.7554/eLife.22599.025
Figure 5—figure supplement 1
Western blot analysis of NHEKs differentiated for 1 day in 1.2 mM Ca++ with Human Keratinocyte Growth Factor (HKGF) supplement to visualize EGFR signaling at different phosphorylation sites upon the loss of Nedd8.
https://doi.org/10.7554/eLife.22599.022
Figure 6
EGFR neddylation is increased in 3D Head and Neck Squamous Cell Carcinoma (HNSCC) and Cops3 knockdown organotypic models.
(a) Biochemical analyses of HNSCC 3D organotypic raft cultures generated using SCC9 and 1483 cell lines displayed a decrease in Dsg1 and Dp, and an increase in EGFR and Cops3. (b) PLA analysis of …
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Figure 6—source data 1
PLA analysis Nedd8/EGFR in 3D cultures.
Quantification of raw data of PLA signal per cell of Nedd8/EGFR in normal skin, and 3D organotypic raft cultures generated using the NHEKs, SCC9s, and 1483 HNSCC cell lines. Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). *p<0.05 (Student’s t test); mean ±SEM (Figure 6c).
- https://doi.org/10.7554/eLife.22599.027
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Figure 6—source data 2
PLA analysis of Nedd8/EGFR 3D organotypic deficient in Cops3.
Quantification of raw data of PLA signals of 3D raft cultures generated with NHEKs treated with siRNA scramble sequence (siCONT) or siRNA directed towards Cops3 (siCOPS3). Quantification performed in FIJI of proximal proteins (see sample size and statistical analysis). *p<0.05 (Student’s t test); mean ±SEM (Figure 6d).
- https://doi.org/10.7554/eLife.22599.028
Author response image 1
Author response image 2
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.22599.029
