Dataset Information

CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration.

ABSTRACT: The Cul4-Cdt2 (CRL4(Cdt2)) E3 ubiquitin ligase is a master regulator of cell-cycle progression and genome stability. Despite its central role in the degradation of many cell-cycle regulators, e.g., Cdt1, p21, and Pr-Set7/Set8, little is known about the regulation of its activity. We report that Cdt2 is autoubiquitylated by the CRL4A E3 ubiquitin ligase. Cdt2 is additionally polyubiquitylated and degraded by Cul1-FBXO11 (CRL1(FBXO11)). CRL1(FBXO11)-mediated degradation of Cdt2 stabilizes p21 and Set8, and this is important during the response to TGF-?, with the Set8 induction being important for turning off the activation of Smad2. The migration of epithelial cells is also stimulated by CRL1(FBXO11)-mediated downregulation of Cdt2 and the consequent stabilization of Set8. This is an interesting example of cross-regulation between specific Cullin 4 and Cullin 1 E3 ubiquitin ligases and highlights the role of ubiquitylation in regulating cellular responses to TGF-? and the migration of epithelial cells.

SUBMITTER: Abbas T

PROVIDER: S-EPMC3615078 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration.

Abbas Tarek T Mueller Adam C AC Shibata Etsuko E Keaton Mignon M Rossi Mario M Dutta Anindya A

Molecular cell 20130307 6

The Cul4-Cdt2 (CRL4(Cdt2)) E3 ubiquitin ligase is a master regulator of cell-cycle progression and genome stability. Despite its central role in the degradation of many cell-cycle regulators, e.g., Cdt1, p21, and Pr-Set7/Set8, little is known about the regulation of its activity. We report that Cdt2 is autoubiquitylated by the CRL4A E3 ubiquitin ligase. Cdt2 is additionally polyubiquitylated and degraded by Cul1-FBXO11 (CRL1(FBXO11)). CRL1(FBXO11)-mediated degradation of Cdt2 stabilizes p21 and ...[more]

PMID: 23478445

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Project description:UNLABELLED:The cullin-based CRL4-CDT2 ubiquitin ligase is emerging as a master regulator of cell proliferation. CRL4-CDT2 prevents re-initiation of DNA replication during the same cell cycle "rereplication" through targeted degradation of CDT1, SET8 and p21 during S-phase of the cell cycle. We show that CDT2 is overexpressed in cutaneous melanoma and predicts poor overall and disease-free survival. CDT2 ablation inhibited a panel of melanoma cell lines through the induction of SET8- and p21-dependent DNA rereplication and senescence. Pevonedistat (MLN4924), a specific inhibitor of the NEDD8 activating enzyme (NAE), inhibits the activity of cullin E3 ligases, thereby stabilizing a vast number of cullin substrates and resulting in cancer cell inhibition in vitro and tumor suppression in nude mice. We demonstrate that pevonedistat is effective at inhibiting the proliferation of melanoma cell lines in vitro through the induction of rereplication-dependent permanent growth arrest as well as through a transient, non-rereplication-dependent mechanism. CRISPR/Cas9-mediated heterozygous deletion of CDKN1A (encoding p21) or SET8 in melanoma cells demonstrated that the rereplication-mediated cytotoxicity of pevonedistat is mediated through preventing the degradation of p21 and SET8 and is essential for melanoma suppression in nude mice. By contrast, pevonedistat-induced transient growth suppression was independent of p21 or SET8, and insufficient to inhibit tumor growth in vivo. Pevonedistat additionally synergized with the BRAF kinase inhibitor PLX4720 to inhibit BRAF melanoma, and suppressed PLX4720-resistant melanoma cells. These findings demonstrate that the CRL4-CDT2-SET8/p21 degradation axis is the primary target of inhibition by pevonedistat in melanoma and suggest that a broad patient population may benefit from pevonedistat therapy. RESEARCH IN CONTEXT:The identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of melanoma. This study identifies CDT2, a substrate receptor for the CRL4 ubiquitin ligase, as a prognostic marker and therapeutic target in melanoma. CDT2 is required for melanoma cell proliferation and inhibition of CRL4(CDT2) by pevonedistat suppresses melanoma in vitro and in vivo through the induction of DNA rereplication and senescence through the stabilization of the CRL4(CDT2) substrates p21 and SET8. Pevonedistat also synergizes with vemurafenib in vivo and suppresses vemurafenib-resistant melanoma cells. These findings show a significant promise for targeting CRL4(CDT2) therapeutically.

Project description:E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix-loop-helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

Dataset Information

CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration.

Publications

CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration.

Similar Datasets

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