👤 Danielle De Bourcier

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

DUSP6/MKP-3 is a cytoplasmic dual-specificity phosphatase specific for the MAP kinases ERK1/2. Previous data have shown that the MEK/ERK axis exerts a retro-control on its own signaling through transcriptional and post-translational regulation of DUSP6. We first confirm the key role of MEK/ERK in maintaining the levels of dusp6 mRNA, while PI3K/mTOR, p38 MAPK, and JNK signaling pathways had no significant effects. We further show that regulation of dusp6 mRNA stability plays a critical role in ERK-dependent regulation of dusp6 expression. Luciferase reporter constructs indicated that MEK/ERK signaling increased the half-life of dusp6 mRNA in a 3'untranslated region (3'UTR)-dependent manner. In addition, hypoxia, a hallmark of tumor growth, was found to increase both endogenous levels of dusp6 mRNA and the stability of the luciferase reporter constructs containing its 3'UTR, in a HIF-1-dependent manner. Nevertheless, a basal ERK activity was required for the response to hypoxia. Finally, Tristetraprolin (TTP), a member of the TIS11 CCCH zinc finger protein family, and PUM2, an homolog of drosophila pumilio, two proteins regulating mRNA stability reduced the levels of endogenous dusp6 mRNA and the activity of the dusp6/3'UTR luciferase reporter constructs. This study shows that post-transcriptional regulation is a key process in the control of DUSP6 expression. Show less