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USP15 is a deubiquitylating enzyme for receptor-activated SMADs

Nature Cell Biology volume 13, pages 1368–1375 (2011)Cite this article

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Abstract

The TGFβ pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGFβ and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition1,2. Several ubiquitin ligases serve as inhibitors of R-SMADs3,4, yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation5. Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGFβ and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGFβ biology.

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Figure 1: USP15 is a DUB required for TGFβ and BMP gene responses.
Figure 2: USP15 is required for TGFβ and BMP biological effects.
Figure 3: USP15 is a DUB for R-SMADs.
Figure 4: R-SMAD activation and nuclear entry promote monoubiquitylation.
Figure 5: USP15-dependent regulative SMAD3 ubiquitylation inhibits DNA binding.

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Acknowledgements

We thank W. Dubiel, P. ten Dijke, C. Hill, A. Moustakas and E. De Robertis for gifts of reagents, and E. Maspero and G. Fontemaggi for help with enzyme purification and ChIP. We are grateful to M. Aragona, F. Zanconato and O. Wessely for comments on the manuscript. This work is supported by grants from the following agencies: AIRC (Italian Association for Cancer Research) PI and AIRC Special Program Molecular Clinical Oncology ‘5 per mille’, University of Padua strategic grant, excellence grant IIT and Comitato Promotore Telethon grant to S.P., and AIRC and MIUR grants to S.D. and M.C. M.I. was a recipient of a Toyobo and Uehara Memorial Foundations grant and is a Marie Curie FP-7 IIF fellow.

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Authors and Affiliations

  1. Department of Medical Biotechnologies, Section of Histology and Embryology, University of Padua, viale G. Colombo 3, 35100 Padua, Italy

    Masafumi Inui, Andrea Manfrin, Anant Mamidi, Graziano Martello, Leonardo Morsut, Sandra Soligo, Elena Enzo, Sirio Dupont, Michelangelo Cordenonsi & Stefano Piccolo

  2. Department of Pharmaceutical Sciences, Molecular Modelling Section, University of Padua, 35100 Padua, Italy

    Stefano Moro

  3. IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Istituto Europeo di Oncologia, 20139 Milan, Italy

    Simona Polo

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  1. Masafumi Inui
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Contributions

M.I. and S.P. designed research; M.I. and A. Manfrin carried out all the biochemical and functional assays in cells and Xenopus embryos. A. Mamidi and S.D. purified SMADs and carried out in vitro binding assays; G.M., S.S. and M.C. helped with Xenopus assays; L.M. and E.E. helped with mesenchymal stem cells and immunofluorescence; S.M. carried out the modelling analysis; S.P. prepared recombinant E3 ligases; S.P. coordinated the work; M.I. and S.P. wrote the manuscript.

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Correspondence to Stefano Piccolo.

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Inui, M., Manfrin, A., Mamidi, A. et al. USP15 is a deubiquitylating enzyme for receptor-activated SMADs. Nat Cell Biol 13, 1368–1375 (2011). https://doi.org/10.1038/ncb2346

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