Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer

Courivaud, Thomas; Ferrand, Nathalie; El-Khattouti, Abdelouahid; Kumar, Santosh; Lévy, Laurence; Ferrigno, Olivier; Atfi, Azeddine; Prunier, Céline

doi:10.1074/jbc.m115.642314

Cited by 16 publications

(20 citation statements)

References 32 publications

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“…We speculate that mutating the loop residues into alanine might affect the relative positioning of critical residues which could be involved in the E2-E3 transthiolation step thus altering the UBE3C E3 ligase enzymatic activity. Functional analysis of WWP1 E3 ligase (NEDD4 HECT subfamily) showed that mutation of its conserved glutamic acid to valine (E798V) substantially increased its autoubiquitination activity in human prostate cancer [43]. The UBE3C HECT E3 ligase has an N-lobe glutamine residue (Q961) in the equivalent position, and we show that mutations of Gln961 (Q961A and Q961E) significantly reduce autoubiquitination activity ( Figure 6D,E).…”

Section: Discussionmentioning

confidence: 69%

Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Singh

Sivaraman

2020

Biochemical Journal

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The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the 'other'. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the 'other' subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744-1083) with an additional fifty N-terminal amino acids (aa 693-743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693-743) preceding the UBE3C HECT domain as well as a loop region (aa 758-762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2-E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

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Section: Discussionmentioning

confidence: 69%

Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Singh

Sivaraman

2020

Biochemical Journal

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“…By analogy with the crystal structure of the related WWP1 HECT, which shows a compact and inactive conformation, this arginine is predicted to lie at the interface between the N lobe and C lobe (electronic supplementary material, figure S1). A natural mutation of WWP1 found in cancer cells maps to a similar interface location, though on an adjacent helix, and has been shown to cause constitutive activation of activity [ 32 ], perhaps by destabilizing this inactive conformation. As expected from this, we found that the arginine mutations in WWP2 partially relieved its autoinhibition, rendering it less dependent on Dvl2.…”

Section: Resultsmentioning

confidence: 99%

Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled

et al. 2015

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Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1–3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state.

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“…KLF2, which includes 355 amino acids, has an automatic suppression region in its first 110-167 amino acids that is capable of binding to the ubiquitin ligase WW domain. This domain contains E3 ubiquitin protein ligase 1 (WWP1), and binding leads to KLF2 protein ubiquitination and degradation [19, 20]. Studies indicate that KLF2 can activate endothelial inflammation by upregulating the expression of endothelial nitric oxides (eNOs) through the NF-kB pathway [21, 22].…”

Section: Introductionmentioning

confidence: 99%

Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2

Guo

Song

Zhao

et al. 2018

Cell Physiol Biochem

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Background/Aims: Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Krüppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism. Methods: We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism. Results: Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the expression of KLF2, mediating VEGF and VEGFR2. Conclusions: Overexpression of AX2R contributes to the inhibition of NV via suppressing KLF2 ubiquitin-dependent protein degradation, which might therefore be a therapeutic option for NV. It could be considered more broadly as an anti-angiogenic agent in the treatment of neovascular-related diseases in the future.

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Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer

Cited by 16 publications

References 32 publications

Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled

Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2

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