UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling
- Université Côte d'Azur, CNRS, IPMC, France
- Université Côte d’Azur, Inserm, C3M, Team Targeting prostate cancer cell metabolism, France
- Université Côte d’Azur, Inserm, C3M, Team Metabolism and cancer, France
- Biomedical Department, Centre Scientifique de Monaco, Monaco
- Biochemistry Laboratory, University Hospital, France
- MRC Centre for Environment and Health, Department of Epidemiology and Biostatistics, School of Public Health, Imperial CollegeLondon, United Kingdom
- Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Sweden
- Inserm U1065, Université Côte d’Azur, France
- CCMA, UFR Sciences, Université Côte d'Azur, France
- Université Côte d’Azur, Inserm, C3M, Team Cellular and Molecular Pathophysiology of Obesity and Diabetes, France
Figures
Figure 1 with 1 supplement
UBTD1 depletion exacerbates EGFR signaling to induce cell proliferation.
(A–G) DU145 cells were transfected for 48 hr with the indicated siRNA (siCTRLpool or siUBTD1pool). (A, D, G) Heatmap of protein phosphorylation relative quantification from multiple signaling …
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Figure 1—source data 1
Uncropped western blot for Figure 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig1-data1-v2.pdf
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Figure 1—source data 2
Row data for Figure 1 and for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
UBTD1 depletion exacerbates EGFR signaling.
(A–L) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or siCTRLsingle1; control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2). (A,C,H) Western …
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Figure 1—figure supplement 1—source data 1
Uncropped western blot for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig1-figsupp1-data1-v2.pdf
Figure 2 with 1 supplement
UBTD1 depletion induces EGFR self-phosphorylation by modifying membrane lipid composition.
(A–F) DU145 cells were transfected for 48 hr with the indicated siRNA (siCTRLpool or siUBTD1pool). (A,B) Heatmap of all (A) lipids and (B) ceramide levels. The normalized expression of each lipid is …
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Figure 2—source data 1
Uncropped western blot for Figure 2.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig2-data1-v2.pdf
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Figure 2—source data 2
Row data for Figure 2 and for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Protein clustering and functional interaction analysis of the UBTD1 interactome.
(A) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or UBTD1: siUBTD1pool). Quantification of lipid class levels, as measured by lipidomics. Data quantification …
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Figure 2—figure supplement 1—source data 1
Uncropped western blot for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig2-figsupp1-data1-v2.pdf
Figure 3
UBTD1 controls ASAH1 ubiquitination to promote EGFR self-phosphorylation.
(A–D) DU145 cells were transfected for 48 hr with the indicated siRNA (siCTRLpool or siUBTD1pool or siUBTD1single1 or single2 or siASAH1 single1 or single2). (A) Immunoblot and quantification of …
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Figure 3—source data 1
Uncropped western blot for Figure 3.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig3-data1-v2.pdf
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Figure 3—source data 2
Row data for Figure 3.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig3-data2-v2.xlsx
Figure 4 with 2 supplements
UBTD1 depletion slows-down EGFR degradation.
(A–F) DU145 cells were transfected for 48 hr with the indicated siRNA (siCTRLpool or siUBTD1pool). (A) Immunoblots (up) and quantification (down) of EGFR levels in cells treated with cycloheximide …
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Figure 4—source data 1
Uncropped western blot for Figure 4.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig4-data1-v2.pdf
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Figure 4—source data 2
Row data for Figure 4 and for Figure 4—figure supplements 1–2.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
UBTD1 depletion slows down EGFR degradation.
(A–K) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or siCTRLsingle1; control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2). (A) Immunoblots (up) …
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Figure 4—figure supplement 1—source data 1
Uncropped western blot for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig4-figsupp1-data1-v2.pdf
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Figure 4—figure supplement 1—source data 2
Uncropped western blot for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig4-figsupp1-data2-v2.pdf
Figure 4—figure supplement 2
UBTD1 depletion slows down RTK degradation.
(A–E) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or UBTD1: siUBTD1pool). (A) HGF-alexa647 pulse chase images and quantification. (B) Proximal ligation assay …
Figure 5 with 1 supplement
UBTD1 interacts with p62/SQSTM1 and controls endolysosomal vesicle positioning.
(A) UBTD1 protein interactome in DU145 cell line centered on EGFR (first neighbors). Node color scale (from blue to red) illustrates the degree (number of interactors). The edges (connecting lines) …
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Figure 5—source data 1
Uncropped western blot for Figure 5.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig5-data1-v2.pdf
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Figure 5—source data 2
Row data for Figure 5 and for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
UBTD1 controls p62/SQSTM1 ubiquitination and endolysosomal vesicles positioning.
(A,B) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or UBTD1: siUBTD1pool). Representative confocal immunofluorescence images (left) and quantification (right) …
Figure 6 with 1 supplement
UBTD1 controls p62/SQSTM1 ubiquitination.
(A–F) DU145 cells were transfected for 48 hr with the indicated siRNA (siCTRLpool or siUBTD1pool or siUBTD1single1 or single2 or RNF26: siRNF26pool). (A) Immunoblots show p62 ubiquitylation in HEK …
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Figure 6—source data 1
Uncropped western blot for Figure 6.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig6-data1-v2.pdf
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Figure 6—source data 2
Raw data for Figure 6 and for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
UBTD1 controls p62/SQSTM1 ubiquitination.
(A–D) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or UBTD1: siUBTD1pool). (A) Immunoblot and quantification of p62. The immunoblot of UBTD1 shows the level …
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Figure 6—figure supplement 1—source data 1
Uncropped western blot for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/68348/elife-68348-fig6-figsupp1-data1-v2.pdf
Author response image 1
Staining of surface EGFR using an antibody against the extracellular region of EGFR bound at 4°C in unpermeabilised.
Control and UBTD1-depleted cells.
Author response image 2
Cells were treated with EGF-647 (100 ng/ml) and stained with a Trans-Golgi network marker (TGN46) to evaluate recycling of EGF-647 in control and UBTD1-depleted cells.
Author response image 3
GM3 levels are increased upon siASAH1 in the DU145 cells.
Additional files
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Supplementary file 1
Proteins identified by LC-MS/MS following UBTD1 immunoprecipitation in DU145 cell line.
- https://cdn.elifesciences.org/articles/68348/elife-68348-supp1-v2.docx
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Supplementary file 2
Subcellular localization score of UBTD1 interactants in DU145 cell line.
- https://cdn.elifesciences.org/articles/68348/elife-68348-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68348/elife-68348-transrepform-v2.pdf
