Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling

Abstract

Proteolytic cleavage and release from the cell surface of membrane-tethered ligands is an important mechanism of regulating intercellular signalling. TACE is a major shedding protease, responsible for the liberation of the inflammatory cytokine TNFα and ligands of the epidermal growth factor receptor. iRhoms, catalytically inactive members of the rhomboid-like superfamily, have been shown to control the ER-to-Golgi transport and maturation of TACE. Here, we reveal that iRhom2 remains associated with TACE throughout the secretory pathway, and is stabilised at the cell surface by this interaction. At the plasma membrane, ERK1/2-mediated phosphorylation and 14-3-3 protein binding of the cytoplasmic amino-terminus of iRhom2 alter its interaction with mature TACE, thereby licensing its proteolytic activity. We show that this molecular mechanism is responsible for triggering inflammatory responses in primary mouse macrophages. Overall, iRhom2 binds to TACE throughout its lifecycle, implying that iRhom2 is a primary regulator of stimulated cytokine and growth factor signalling.

Article and author information

Author details

  1. Adam Graham Grieve

    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  2. Hongmei Xu

    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  3. Ulrike Künzel

    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  4. Paul Bambrough

    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  5. Boris Sieber

    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  6. Matthew Freeman

    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
    For correspondence
    matthew.freeman@path.ox.ac.uk
    Competing interests
    Matthew Freeman, Reviewing editor, <i>eLife</i>.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0410-5451

Funding

Wellcome (101035/Z/13/Z)

  • Adam Graham Grieve
  • Hongmei Xu
  • Ulrike Künzel
  • Paul Bambrough
  • Boris Sieber
  • Matthew Freeman

Medical Research Council (Graduate student scholarship)

  • Ulrike Künzel

Boehringer Ingelheim Fonds (Graduate student scholarship)

  • Ulrike Künzel
  • Boris Sieber

National Natural Science Foundation of China (31640023)

  • Hongmei Xu

Horizon 2020 Framework Programme (Marie Sklodowska-Curie grant agreement 659166)

  • Adam Graham Grieve

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

Ethics

Animal experimentation: This study was performed in strict accordance with University of Oxford and UK Government rules and guidelines. The procedures and justification for the research was approved under UK PPL 80/2584.

Copyright

© 2017, Grieve 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.

Metrics

  • 3,331
    views
  • 654
    downloads
  • 88
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Adam Graham Grieve
  2. Hongmei Xu
  3. Ulrike Künzel
  4. Paul Bambrough
  5. Boris Sieber
  6. Matthew Freeman
(2017)
Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling
eLife 6:e23968.
https://doi.org/10.7554/eLife.23968

Share this article

https://doi.org/10.7554/eLife.23968

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Senem Ntourmas, Martin Sachs ... Dominic B Bernkopf
    Research Article

    Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

    1. Biochemistry and Chemical Biology
    2. Physics of Living Systems
    Debabrata Dey, Shir Marciano ... Gideon Schreiber
    Research Article

    For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between pKa and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated in the cell cytoplasm. Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant. Our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules. Surprisingly, blocking lysosomal import only slightly increased diffusion rates and fractional recovery. Conversely, blocking protonation by N-acetylated analogues, greatly enhanced their diffusion and fractional recovery after FRAP. Based on these results, N-acetylation of small molecule drugs may improve the intracellular availability and distribution of weakly basic, small molecule drugs within cells.