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Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGF? signalling and EMT.

ABSTRACT: Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGF?-dependent biological outputs such as epithelial-mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGF? pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGF?-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGF?-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGF?-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGF? stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads.

SUBMITTER: Soond SM

PROVIDER: S-EPMC4073228 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT.

Soond S M SM Chantry A A

Oncogene 20110124 21

Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGFβ-dependent biological outputs such as epithelial-mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGFβ pathway. Interesting ...[more]

PMID: 21258410

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Project description:BackgroundTransitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFβ signaling.MethodsNucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments.Resultsrs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140.ConclusionsAs far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFβ signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.

Dataset Information

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGF? signalling and EMT.

Publications

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT.

Similar Datasets

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