👤 S M Soond

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein-protein interaction WW domains. The WW4 domain-containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial-mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3-WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis. Show less

The WWP2 E3 ubiquitin ligase has previously been shown to regulate TGFβ/Smad signalling activity linked to epithelial-mesenchymal transition (EMT). Whilst inhibitory I-Smad7 was found to be the preferred substrate for full-length WWP2-FL and a WWP2-C isoform, WWP2-FL also formed a stable complex with an N-terminal WWP2 isoform (WWP2-N) in the absence of TGFβ, and rapidly stimulated activating Smad2/3 turnover. Here, using stable knockdown experiments we show that specific depletion of individual WWP2 isoforms impacts differentially on Smad protein levels, and in WWP2-N knockdown cells we unexpectedly find spontaneous expression of the EMT marker vimentin. Re-introduction of WWP2-N into WWP2-N knockout cells also repressed TGFβ-induced vimentin expression. In support of the unique role for WWP2-N in regulating TGFβ/Smad functional activity, we then show that a novel V717M-WWP2 mutant in the MZ7-mel melanoma cell line forms a stable complex with the WWP2-N isoform and promotes EMT by stabilizing Smad3 protein levels. Finally, we report the first analysis of WWP2 expression in cancer cDNA panel arrays using WWP2 isoform-specific probes and identify unique patterns of WWP2 isoform abundance associated with early/advanced disease stages. WWP2-N is significantly downregulated in stage IIIC melanoma and up-regulated in stage II/III prostate cancer, and we also find isolated examples of WWP2-FL and WWP2-C overexpression in early-stage breast cancer. Together, these data suggest that individual WWP2 isoforms, and particularly WWP2-N, could play central roles in tumourigenesis linked to aberrant TGFβ-dependent signalling function, and also have potential as both prognostic markers and molecular therapeutic targets. Show less

Ubiquitination of protein species in regulating signal transduction pathways is universally accepted as of fundamental importance for normal development, and defects in this process have been implicated in the progression of many human diseases. One pathway that has received much attention in this context is transforming growth factor-beta (TGF-β) signalling, particularly during the regulation of epithelial-mesenchymal transition (EMT) and tumour progression. While E3-ubiquitin ligases offer themselves as potential therapeutic targets, much remains to be unveiled regarding mechanisms that culminate in their regulation. With this in mind, the focus of this review highlights the regulation of the ubiquitination pathway and the significance of a recently described group of NEDD4 E3-ubiquitin ligase isoforms in the context of TGF-β pathway regulation. Moreover, we now broaden these observations to incorporate a growing number of protein isoforms within the ubiquitin ligase superfamily as a whole, and discuss their relevance in defining a new 'iso-ubiquitinome'. Show less

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. 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 TGFβ-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 TGFβ-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGFβ-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 TGFβ 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. Show less