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Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms

Lloyd C. Wahl, Jessica E. Watt, Hiu T. T. Yim, Danielle De Bourcier, James Tolchard, Surinder M. Soond, Tharin M. A. Blumenschein, Andrew Chantry
Published: 21 September 2019
 by  MDPI
International Journal of Molecular Sciences , Volume 20; doi:10.3390/ijms20194682

Abstract: 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.
Keywords: Smad / Smad7 / protein interaction / transforming growth factor beta / Nedd4 / E3 ubiquitin ligase / WW domain / TGFβ signalling

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