Multiple Cullin-Associated E3 Ligases Regulate Cyclin D1 Protein Stability

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    This paper is of interest to cell biologists studying the mechanisms of protein posttranslational modifications. The study investigates Cullin-RING ubiquitin E3 ligases (CRLs) that regulate cyclin D1 protein stability in cells by utilizing siRNA screening and ectopic expression approaches. By screening a siRNA library containing different E3 ligases, the authors identified a previously uncharacterized combination of Cullin 1-7 and associated E3 ligases (Keap1-CUL3, DDB2-CUL4A/4B, WSB2-CUL2/5, and RBX1-CUL1-7) that are important for cyclin D1 ubiquitination and proteasome-mediated degradation.

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Abstract

Cyclin D1 is a key regulator of cell cycle progression, which forms a complex with CDK4/6 to regulate G1/S phase transition during cell cycle progression. Cyclin D1 has been recognized as an oncogene since it was upregulated in several different types of cancers. It is known that the post-translational regulation of cyclin D1 is controlled by ubiquitination/ proteasome degradation system in a phosphorylation-dependent manner. A few cullin-associated F-box E3 ligases have been shown to regulate cyclin D1 degradation; however, it is not known if additional cullin-associated E3 ligases participate in the regulation of cyclin D1 protein stability. In this study, we have screened a siRNA library containing siRNAs specific for 154 ligase subunits, including F-box, SOCS, BTB-containing proteins and DDB proteins. We found that multiple cullin-associated E3 ligases regulate cyclin D1 activity, including Keap1, DDB2, WSB2 and Rbx1. We found that these E3 ligases directly interact with cyclin D1, regulate cyclin D1 ubiquitination and proteasome degradation in a phosphorylation-dependent manner. These E3 ligases also control cell cycle progression and cell proliferation through regulation of cyclin D1 protein stability. Our study provides novel insights into regulatory mechanisms of cyclin D1 protein stability and function.

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  1. Author Response

    Reviewer #2 (Public Review):

    Major weaknesses:

    1. The biggest weakness of the manuscript is the lack of appropriate explanation and interpretation of these observed cyclin D1 ubiquitination and degradation by at least five different combinations of Cullin-E3 ligases. Are all the five cullin-E3 combinations exclusive and/or redundant to each other for cyclin D1 ubiquitination? What are the speculations in terms of the underlying mechanism? At least a working model should be included to better interpret the data.

    Cyclin D1 has been recognized as an oncogene, which is upregulated in multiple types of cancers. In different types of cells, different E3 ligase may be involved in the process of cyclin D1 protein degradation. Even in the same cells, multiple E3 ligases may be involved in cyclin D1 degradation to make sure that steady-state protein levels of cyclin D1 are under surveillance and fine-tune regulation.

    1. Although a phosphorylation-mutant cyclin D1 (i.e., T286) was included in the manuscript, there is no Lysine residue mutant within cyclin D1 identified and characterized for the critical function of cyclin D1 ubiquitination.

    It was reported that Lysine 269 is essential for cyclin D1 ubiquitination (Barbash et al., 2009). 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 levels were detected by the Western blot analysis. We found the expression of WT cyclin D1 was decreased in HEK293 cells with Keap1, DDB2, and AMBRA1 co-transfected, while the expression of K269R mutant cyclin D1 showed no significant decrease in rhe cells co-transfected with co-transfected Keap1, DDB2, and AMBRA1, suggesting that Lysine 269 is essential for cyclin D1 ubiquitination.

    1. The significance of these different Cullin 1-7 and associated E3 ligases (Keap1-CUL3, DDB2-CUL4A/4B, WSB2-CUL2/5, and RBX1-CUL1-7) in cyclin D1 ubiquitination is mainly determined by siRNA-mediated knockdown or overexpression of target cullin/E3 proteins. However, it is not clear whether the observed phenotypes of cyclin D1 are due to these cullin-E3 ligases directly or indirectly. In vitro ubiquitination assay with E1, E2, and E3 should be performed to demonstrate whether recombinant cyclin D1 is ubiquitinated.

    We have performed in vitro ubiquitination assay as the reviewer suggested. The results demonstrated that Keap1, DDB2, and WSB2 can induce cyclin D1 ubiquitination. Especially, Keap1 induced cyclin D1 ubiquitination and formed ubiquitination ladder similar to AMBRA1-induced cyclin D1 ubiquitination ladder. In contrast, no clear ubiquitination ladder was observed in Rbx1 group (Figure S16).

  2. eLife assessment

    This paper is of interest to cell biologists studying the mechanisms of protein posttranslational modifications. The study investigates Cullin-RING ubiquitin E3 ligases (CRLs) that regulate cyclin D1 protein stability in cells by utilizing siRNA screening and ectopic expression approaches. By screening a siRNA library containing different E3 ligases, the authors identified a previously uncharacterized combination of Cullin 1-7 and associated E3 ligases (Keap1-CUL3, DDB2-CUL4A/4B, WSB2-CUL2/5, and RBX1-CUL1-7) that are important for cyclin D1 ubiquitination and proteasome-mediated degradation.

  3. Reviewer #1 (Public Review):

    As central molecular scaffolds, Cullin ring ubiquitin ligases proteins play critical roles in the post-translational modification of cellular proteins. Since cyclin D1 is a pivotal regulator to form the CDK4/6 complex during cell cycle progression, understanding if additional cullin-associated E3 ligases participate in the regulation of cyclin D1 protein stability is interesting. The current study used an NIH3T3 cells-based siRNA library to screen 156 cullin-associated ubiquitin ligases genes. The results indicated that cullins are required for cyclin D1 degradation, and cullin-induced cyclin D1 degradation is ubiquitin-dependent and is mediated by multiple E3 ligases (Keap1, DDB2, WSB2, and Rbx1 subunits). Overall, this is a well-designed experimental study and the quality of the data collection and analysis are high and rigorous. The manuscript is well written. The conclusion stated by the authors is supported by their data logically.

  4. Reviewer #2 (Public Review):

    In this manuscript, Lu et al. aimed to identify and characterize how cyclin D1 is ubiquitinated and degraded through Cullin-E3 ligases in addition to the well-documented CUL1/7-F-box proteins (Fbxw8, Fbx4, and Fbx031). The authors first confirmed that in addition to CUL1/7, all seven Cullin proteins (CUL1, 2,3, 4A, 4B, 5, and 7) are required for cyclin D1 degradation via overexpression or siRNA-mediated knockdown approach. Next, these seven Cullin proteins are validated as critical factors for cyclin D1 ubiquitination and proteosome-mediated degradation via a phosphorylation-dependent mechanism. A siRNA library of 154 e3 ligases was screened to identify 24 E3 ligases for cyclin D1 degradation, five of which (Fbxw8, Keap1, DDB2, WSB2, and Rbx1) were selected for further analysis. Functional validation and characterization analyses have shown that Keap1, DDB2, WSB2, and Rbx1 interact with cyclin D1, and that Wild Type but not mutant version of Keap1, DDB2, WSB2, and Rbx1 induces cyclin D1 ubiquitination and degradation. Finally, these cullin-E3 ligases-mediated cyclin D1 degradation is important for cell cycle progression, DNA replication, and cell viability.

    Although the experimental design is overall sound and the presentation of the data is great, some major weaknesses (see details below) dampen the rigor of the study and overall claim.

    Major weaknesses:

    1. The biggest weakness of the manuscript is the lack of appropriate explanation and interpretation of these observed cyclin D1 ubiquitination and degradation by at least five different combinations of Cullin-E3 ligases. Are all the five cullin-E3 combinations exclusive and/or redundant to each other for cyclin D1 ubiquitination? What are the speculations in terms of the underlying mechanism? At least a working model should be included to better interpret the data.

    2. Although a phosphorylation-mutant cyclin D1 (i.e., T286) was included in the manuscript, there is no Lysine residue mutant within cyclin D1 identified and characterized for the critical function of cyclin D1 ubiquitination.

    3. The significance of these different Cullin 1-7 and associated E3 ligases (Keap1-CUL3, DDB2-CUL4A/4B, WSB2-CUL2/5, and RBX1-CUL1-7) in cyclin D1 ubiquitination is mainly determined by siRNA-mediated knockdown or overexpression of target cullin/E3 proteins. However, it is not clear whether the observed phenotypes of cyclin D1 are due to these cullin-E3 ligases directly or indirectly. In vitro ubiquitination assay with E1, E2, and E3 should be performed to demonstrate whether recombinant cyclin D1 is ubiquitinated.

  5. Reviewer #3 (Public Review):

    Lu, Zhang et al. utilize siRNA-mediated depletion and ectopic expression to show that CUL1-7, the scaffold proteins of CRLs, control levels of ectopically expressed cyclin D1, but not a phosphorylation deficient cyclin D1 variant (T286A) in HEK293 cells. This process occurs in a proteasome-dependent manner. Through an siRNA screen for CRL substrate adaptors in NIH3T3 cells, using a previously established Cyclin D1 activity reporter, the authors then identify the CRL adaptors KEAP1 (CRL3), DDB2 (CRL4A/B), and WSB2 (CRL2/5) as new candidate regulators of cyclin D1. They provide evidence that these CRL substrate adaptors, when ectopically expressed, co-immunoprecipitate with endogenous cyclin D1 and induce ubiquitylation and proteasomal degradation of ectopically expressed cyclin D1 in HEK293 cells. In addition, through siRNA depletion and CHX chase assays, the authors provide evidence that KEAP1, DDB2, and WSB2 are regulating the half-life of endogenous cyclin D1 in HEK293 cells. Finally, experiments in HCT-116 cells that ectopic expression of KEAP1, DDB2, and WSB2, inhibit cell growth in cells stably expressing exogenous cyclin D1, but not a phosphorylation deficient cyclin D1 variant (T286A). From these results, the authors conclude that cyclin D1 degradation in cells is mediated by multiple CRLs.

    Strength:
    This study identifies new candidate regulators of cyclin D1 protein levels KEAP1, DDB2, and WSB2.

    Weaknesses:
    While this study provides evidence that KEAP1, DDB2, and WSB2 are candidate regulators of cyclin D1 protein levels, the co-IP experiments and CHX chases lack important controls or are not convincing. More importantly, there are no experiments demonstrating that cyclin D1 is directly ubiquitylated by these substrate adaptors in the context of their respective CRL complexes, the main conclusion of this short report. Another major weakness is the omission of recent studies that demonstrate that the major E3 ligase degrading cyclin D(1-3) is CRL4-AMBRA1 (Simoneschi et al., Nature 2021; Maiani et al., Nature 2021; Chaikovsky et al., Nature 2021). In these studies, three independent groups taking complementary approaches show that in several cell lines and contexts CRL4-AMBRA1 is the only ligase degrading cyclin D and other cullins and substrate adaptors have little to no effect. While these data do not rule out the existence of other CRLs regulating cyclin D, they raise the question of under which conditions and in which cell lines other CRLs would be important for cyclin D degradation, a question that is not addressed or discussed.