Multiple cullin-associated E3 ligases regulate cyclin D1 protein stability
- Research Center for Computer-aided Drug Discovery, Chinese Academy of Sciences, China
- Department of Oncology, Johns Hopkins University, United States
- Department of Biochemistry, Southern University of Science and Technology, China
Figures
Figure 1 with 12 supplements
Cullin-associated E3 ligases mediate cyclin D1 ubiquitination and proteasome degradation.
(A) Co-immunoprecipitation (co-IP) of Keap1, CUL3 with endogenous cyclin D1. HA-Keap1 and Myc-CUL3 were co-transfected into HEK293 cells with the MG132 treatment (10 μM, 4 hr incubation). 24 hr …
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Figure 1—source data 1
Numerical data obtained during experiments represented in Figure 1.
- https://cdn.elifesciences.org/articles/80327/elife-80327-fig1-data1-v2.zip
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Figure 1—source data 2
Original western blot files for Figure 1.
- https://cdn.elifesciences.org/articles/80327/elife-80327-fig1-data2-v2.zip
Figure 1—figure supplement 1
Cyclin D1 degradation is mediated by multiple cullins.
(A) Cullin1, 2, 3, 4A, 4B, 5 and 7 constructs were co-transfected with cyclin D1 into HEK293 cells. Cyclin D1, cyclin B1 and cyclin A protein levels were detected through western blotting. (B) …
Figure 1—figure supplement 2
Silencing efficiency of the cullin siRNAs was determined by the luciferase assay.
Cyclin D1 expression plasmid and 3xE2F-luc reporter construct were co-transfected with specific siRNAs targeting cullin genes into HEK293 cells. Luciferase assay were performed 24h after the …
Figure 1—figure supplement 3
Specificity of cullin 4A and 4B siRNAs.
Control siRNA (C) or siRNAs specific for each cullin (1, 2, 3, 4A, 4B, 5 and 7) were transiently tranfected into HEK293 cells. Western blotting was performed detecting the Cul4A and Cul4B protein …
Figure 1—figure supplement 4
Specificity of cullin siRNAs.
Control siRNA or siRNAs specific for each cullin (1, 2, 3, 4A, 4B, 5 and 7) were transiently tranfected into HEK293 cells. Real-time PCR was performed to detect levels of each cullin mRNA. Data are …
Figure 1—figure supplement 5
Cullins affect cyclin D1 activity.
Cyclin D1 expression plasmid and 3xE2F-luc reporter construct were co-transfected with each cullin iinto HEK293 cells. Luciferase assay were performed at three different time points: 12, 24 and 48 …
Figure 1—figure supplement 6
Silencing of cullins enhances cyclin D1 activity.
Cyclin D1 expression plasmid and 3xE2F-luc reporter construct were co-transfected with each siRNA specific for each cullin into HEK293 cells. Luciferase assay were performed 24 hours after the …
Figure 1—figure supplement 7
Cyclin D1 ubiquitination is mediated by multiple cullins.
WT or mutant cyclin D1 (T286A) expression plasmids were co-transfected with cullins expression plasmids into HEK293 cells with the treatment of proteasome inhibitor MG132 (10 μM, 4h incubation). 24 …
Figure 1—figure supplement 8
Proteasomal inhibitor reverses cullin-mediated cyclin D1 degradation.
Cyclin D1 and cullin expression plasmids were co-transfected into HEK293 cells with or without MG132 treatment (10 μM, 4h incubation). Changes in cyclin D1 protein levels were detected by the …
Figure 1—figure supplement 9
Effects of cullins on cyclin D1 mRNA expression.
HCT-116 cells were transiently transfected with CUL1, 2, 3, 4A, 4B, 5 or 7 respectively. Total RNAs were harvested 24 hours after transfection. Changes in cyclin D1 mRNA expression was detected by …
Figure 1—figure supplement 10
Cullin-associated ubiquitin ligases siRNA library screening.
(A and B) 468 siRNAs targeting 156 genes (triplicate) were coated in 96-well plates (silencer Custom siRNA library, Ambion, Austin, USA). Cyclin D1 expression plasmid and 3xE2F-luc reporter …
Figure 1—figure supplement 11
Five different cullin-associated ubiquitin ligases mediate cyclin D1 degradation.
Cyclin D1 expression plasmid was co-transfected with Fbxw8 (associated with CUL1), Keap1 (associated with CUL3), DDB2 (associated with CUL4A and 4B), WSB2 (associated with CUL2 and 5) and Rbx1 …
Figure 1—figure supplement 12
Silencing effects of Keap1, DDB2, WSB2, and Rbx1 siRNAs are specific to their target genes.
Control (Ctrl) siRNA or siRNAs specific for Keap1, DDB2, WSB2 and Rbx1 were transiently tranfected into HEK293 cells. Expression of Keap1 and KLHL12 (interact with CUL3), DDB2 and DDB1 (interact …
Figure 2 with 4 supplements
Cullin-associated E3 ligases affect cyclin D1 function.
(A, D, G, J) Keap1, DDB2, WSB2, or Rbx1 siRNAs were transiently transfected into human colon cancer cell line HCT-116 cells. Endogenous cyclin D1 protein levels were detected by the western blot …
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Figure 2—source data 1
Numerical data obtained during experiments represented in Figure 2.
- https://cdn.elifesciences.org/articles/80327/elife-80327-fig2-data1-v2.xlsx
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Figure 2—source data 2
Original western blot files for Figure 2.
- https://cdn.elifesciences.org/articles/80327/elife-80327-fig2-data2-v2.zip
Figure 2—figure supplement 1
Fluorescence-activated cell sorting (FACS) analysis.
Keap1 (A), DDB2 (B), WSB2 (C) and Rbx1 (D) expression plasmids were transiently transfected into HCT-116 cells stably transfected with WT or mutant cyclin D1 (T286A). 48 hours after transfection, …
Figure 2—figure supplement 2
Cullin-associated E3 ligases decreased phospho-Rb expression.
WT or mutant cyclin D1 (T286A) expression plasmids were co-transfected with Keap1, WSB2, DDB2 or Rbx1 expression plasmids into HEK293 cells. 48 hours after transfection, changes in phospho-Rb and …
Figure 2—figure supplement 3
Cullin-associated E3 ligases couldn’t degrade K269R mutant cyclin D1.
WT or mutant cyclin D1 (K269R) expression plasmids were co-transfected with Keap1, DDB2, and AMBRA1 expression plasmids into HEK293 cells. 48 hours after transfection, changes in cyclin D1 protein …
Figure 2—figure supplement 4
In vitro ubiquitination assay of cyclin D1 by Keap1, DDB2, WSB2, or AMBRA1.
In vitro ubiquitination of cyclin D1 by different Cullin-associated E3 ligases as indicated. Cyclin D1 and its ubiquitinated forms (bracket indicated) were detected by immunoblotting with an …
