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. 2019 Sep 21;20(19):4682.
doi: 10.3390/ijms20194682.

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

Lloyd C Wahl  1 , Jessica E Watt  2 , Hiu T T Yim  3 , Danielle De Bourcier  4 , James Tolchard  5 , Surinder M Soond  6 , Tharin M A Blumenschein  7 , Andrew Chantry  8
Affiliations

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

Lloyd C Wahl et al. Int J Mol Sci. .

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: E3 ubiquitin ligase; NEDD4; TGFβ signalling; WW domain; protein interaction; smad; smad7; transforming growth factor beta.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The three-stranded anti-parallel β-sheet structure of WWP2 WW4 solved by NMR. (A) A schematic of the B1 domain of streptococcal protein G (GB1)–WW4 recombinant protein. (B) Ribbon diagram depictions of the WWP2 WW4 domain from the most representative model (model 1) of the refined 20-model structural ensemble.
Figure 2
Figure 2
The interaction between WWP2 WW4 and Smad7/S206-phosphorylated Smad7 (pSmad7). (A) The Smad7 ligand, with the PPxY motif in bold and the putative S206 phosphorylation site in red. (B) The superimposed WW4/Smad7 titration heteronuclear single quantum coherence (HSQCs). Lower ligand concentrations are in light grey, and higher ligand concentrations are in dark grey. (C) The superimposed WW4/pSmad7 titration HSQCs. Lower ligand concentrations are in light blue, and higher ligand concentrations are in dark blue. (D) The WW4/Smad7 (1.19 ± 0.21 mM) and WW4/pSmad7 (0.5 ± 0.07 mM) dissociation constants. (E) The shift distances of the WW4/Smad7 and WW4/pSmad7 titration HSQC amide peaks in ppm. The WW4 secondary structure is aligned to the residue number along the x-axis, with β-strands represented as orange boxes. (F) The WWP2 WW4 domain structure with Smad7 binding site residues colour-coded for titration shift distances. The labelled residues are the XP-binding groove (472Phe and 461Tyr) and the second specificity pocket (463Val, 465His and 468Arg), which determine specificity for the PPxY recognition motif.
Figure 3
Figure 3
Splice variation at the wwp2 gene. (A) A Western blot of HEK293A cells transfected with combinations of mouse and/or human epithelial splicing regulatory proteins (ESRPs) as indicated and stimulated with TGFβ; the membrane was probed with anti-WWP2C antibody or anti-β-actin antibody as a control. (B) The wwp2 gene locus (not to scale) with aligned transcripts and expressed sequence tag (ESTs) as annotated. (C) Semi-quantitative RT-PCR using primers _targeting intron 9/10 of wwp2 mRNA extracted from A375 cells treated with TGFβ over 18 h. (D) Semi-quantitative RT-PCR using primers _targeting exon 17–intron 19/20 of wwp2 mRNA extracted from COLO-357 and VCaP cells treated with TGFβ over 8 h and from A375 and SK-MEL28 cells treated over 6 h. (E) A schematic showing the domain composition of WWP2-FL, WWP2-C and the putative new isoform WWP2C-ΔHECT. (F) Fold change in luciferase activity in HEK293A cells co-transfected with WWP2C-ΔHECT and the Smad3-dependent CAGA12-luciferase reporter, treated and untreated with TGFβ, with standard error bars.
Figure 4
Figure 4
The interaction between tandem WWP2 WW3–4 and Smad7. (A) The superimposed WW3–4/Smad7 titration HSQCs. Lower ligand concentrations are in light orange, and higher ligand concentrations are in dark orange. (B) The superimposed WW3/Smad7 titration HSQCs. Lower ligand concentrations are in light green, and higher ligand concentrations are in dark green. (C) The superimposed WW4/Smad7 titration HSQCs. Lower ligand concentrations are in light purple, and higher ligand concentrations are in dark purple. (D) The shift distances of the WW3–4/Smad7 (orange), WW3/Smad7 (green) and WW4/Smad7 (purple) titration HSQC amide peaks in ppm. The WW3–4 secondary structure is aligned to the residue number along the x-axis, with β-strands represented as yellow boxes. (E) The WW3–4/Smad7 binding curve KD fit. (F) The WW3/Smad7 (139 ± 14.4 µM), WW3(tandem)/Smad7 (20.57 ± 26.24 µM), WW4/Smad7 (237 ± 15.7 µM) and WW4(tandem)/Smad7 (249.3 ± 68.1 µM) dissociation constants. (G) HSQC peaks belonging to the WW3 domain at different chemical shifts for WW3–4 (orange) and WW3 (green).
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