Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2
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Evaluation Summary:
The identification of p130 as a substrate of cyclin F adds a new level of understanding about the role of this ubiquitin ligase in cell cycle control. While much of the data are string and of interest, several concerns need to be addressed with the inclusion of new experimental data. This work will be of interest to researchers in the fields of cell cycle and cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107, and RBL2/p130, coordinately represses cell cycle gene expression, inhibiting proliferation, and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin-dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCF cyclin F . We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCF cyclin F in the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.
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Evaluation Summary:
The identification of p130 as a substrate of cyclin F adds a new level of understanding about the role of this ubiquitin ligase in cell cycle control. While much of the data are string and of interest, several concerns need to be addressed with the inclusion of new experimental data. This work will be of interest to researchers in the fields of cell cycle and cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Enrico and colleagues identify a genetic link between cyclin F and the RB-CDK network using DEPMAP genome-wide datasets with p130 identified as a cyclin F substrate potentially explaining why p130 is degraded in SKP2 KO MEFs. The cyclin F interaction was mapped to an RxL motif on p130, which when mutated prevent proliferation, cell cycle progression, and cell cycle gene expression in NHF-1 cells. However, this RxL motif on p130 was previously implicated in cyclin/CDK complex binding, implicating a deficiency in CDK phosphorylation rather than an increase in protein stability to explain its activity.
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Reviewer #2 (Public Review):
The manuscript provides several lines of evidence to support the conclusion that cyclin F interacts with and regulates the stability of p130. The authors first note the connection between Rb/CDK pathway and cyclin F by analysis of the DepMap cell survival data, and then identify p130 as a candidate substrate of cyclin F. The authors use a series of well-designed experiments to demonstrate that overexpression of cyclin F promotes the degradation of retinoblastoma family member p130 in HEK293T cells, and identify a conserved amino acid sequence 680-RRL-682 in a linker domain of p130 that is responsible for cyclin F recognition and ubiquitination. Mutation of this site in p130 results in an increased protein stability and enhanced ability to suppress cell proliferation by repressing the DREAM target genes. The …
Reviewer #2 (Public Review):
The manuscript provides several lines of evidence to support the conclusion that cyclin F interacts with and regulates the stability of p130. The authors first note the connection between Rb/CDK pathway and cyclin F by analysis of the DepMap cell survival data, and then identify p130 as a candidate substrate of cyclin F. The authors use a series of well-designed experiments to demonstrate that overexpression of cyclin F promotes the degradation of retinoblastoma family member p130 in HEK293T cells, and identify a conserved amino acid sequence 680-RRL-682 in a linker domain of p130 that is responsible for cyclin F recognition and ubiquitination. Mutation of this site in p130 results in an increased protein stability and enhanced ability to suppress cell proliferation by repressing the DREAM target genes. The authors show evidence that this regulation is direct using in vitro ubiquitination assay with a reconstituted cyclin F E3 ligase complex. Studies with ectopically expressed p130 mutant refractory to cyclin F degradation demonstrate that loss of this regulation could significantly inhibit cell proliferation and possibly increase apoptosis, which could be especially relevant for ALS pathogenesis. Overall, the data support the role of cyclin F in regulation of the levels of p130, and increases our understanding of the functional significance of both p130 and cyclin F in control of cell proliferation.
Although the data presented in the manuscript is extensive and convincing, this study could be strengthened by additional supportive evidence that cyclin F is indeed a direct physiological regulator of p130 during the cell cycle progression. The authors demonstrate that depletion of cyclin F causes a robust upregulation of p130 in human fibroblasts cells. Given that cyclin F regulates many substrates involved in cell cycle progression, including activator E2Fs and B-Myb transcription factor, these changes in p130 levels could be caused by changes in the cell cycle status, which was not assessed in this study. Furthermore, major conclusions in this study rely on assays using proteins overexpressed in HEK293T or HeLa cells. Since these cells express viral oncoproteins (SV40 Large T antigen and HPV E7 protein, respectively), which are known to bind Rb family members including p130 and target them to proteasomal degradation, it is possible that some of the observed effects in these cell lines are mediated by viral proteins that could be influenced by cyclin F. For example, additional validation of the interaction between the two proteins in non-virus transformed cell line would strengthen the conclusion the cyclin F is indeed a physiological regulator of p130. Notably, the identity of E3 ligase(s) responsible for p130 degradation by E7 or Large T, is not known, and it would be interesting to see if cyclin F is involved. Furthermore, phosphorylation of p130 by cyclin-CDKs has been shown to play a major role in promoting its cell-cycle dependent degradation by proteasome. The role of phosphorylation in regulation of p130 by cyclin F was not investigated in this study, and it remains to be established whether cyclin F controls the levels of p130 in coordination with, or independent of the cyclin-CDKs. The conclusion that stabilized p130 mutant refractory to cyclin F regulation could promote apoptosis also could be strengthened by providing additional evidence.
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Reviewer #3 (Public Review):
The authors showed that p130 is a substrate of cyclin F. In addition, the RxL motif in the p130 pocket domain mediates binding, ubiquitination and stability. Expression of mutant p130 which can't be ubiquitinated and degraded impairs cell proliferation and cell cycle progression. These data are straightforward.
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