1. Biochemistry and Chemical Biology
  2. Cell Biology
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TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

  1. Anassuya Ramachandran
  2. Pedro Vizan
  3. Debipriya Das
  4. Probir Chakravarty
  5. Janis Vogt
  6. Katherine W Rogers
  7. Patrick Müller
  8. Andrew P Hinck
  9. Gopal P Sapkota
  10. Caroline S Hill  Is a corresponding author
  1. The Francis Crick Institute, United Kingdom
  2. University of Dundee, United Kingdom
  3. Friedrich Miescher Laboratory of the Max Planck Society, Germany
  4. University of Pittsburgh School of Medicine, United States
Research Article
  • Cited 65
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Cite this article as: eLife 2018;7:e31756 doi: 10.7554/eLife.31756

Abstract

The best characterized signaling pathway downstream of the transforming growth factor β (TGF-β) pathway is through SMAD2 and SMAD3. However, TGF-β also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-β-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-β-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-β-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-β-induced transcriptional program and physiological responses.

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Author details

  1. Anassuya Ramachandran

    Developmental Signalling Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Pedro Vizan

    Developmental Signalling Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Debipriya Das

    Developmental Signalling Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Probir Chakravarty

    Bioinformatics and Biostatistics Facility, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Janis Vogt

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Katherine W Rogers

    Systems Biology of Development Group, Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5700-2662
  7. Patrick Müller

    Systems Biology of Development Group, Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0702-6209
  8. Andrew P Hinck

    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Gopal P Sapkota

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9931-3338
  10. Caroline S Hill

    Developmental Signalling Laboratory, The Francis Crick Institute, London, United Kingdom
    For correspondence
    caroline.hill@crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8632-0480

Funding

Francis Crick Institute (FC001095)

  • Anassuya Ramachandran
  • Pedro Vizan
  • Debipriya Das
  • Probir Chakravarty
  • Caroline S Hill

NIH Office of the Director (GM58670)

  • Andrew P Hinck

NIH Office of the Director (CA172886)

  • Andrew P Hinck

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Roger J Davis, University of Massachusetts Medical School, United States

Publication history

  1. Received: September 5, 2017
  2. Accepted: January 26, 2018
  3. Accepted Manuscript published: January 29, 2018 (version 1)
  4. Version of Record published: March 1, 2018 (version 2)

Copyright

© 2018, Ramachandran et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

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    A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.