1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

  1. Ruming Chen
  2. Cláudia Rato
  3. Yahui Yan
  4. Ana Crespillo-Casado
  5. Hanna J Clarke
  6. Heather P Harding
  7. Stefan J Marciniak
  8. Randy J Read
  9. David Ron  Is a corresponding author
  1. University of Cambridge, United Kingdom
https://doi.org/10.7554/eLife.04871
Share this article
Cite this article
  1. Ruming Chen
  2. Cláudia Rato
  3. Yahui Yan
  4. Ana Crespillo-Casado
  5. Hanna J Clarke
  6. Heather P Harding
  7. Stefan J Marciniak
  8. Randy J Read
  9. David Ron
(2015)
G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases
eLife 4:e04871.
https://doi.org/10.7554/eLife.04871
  2. Download BibTeX
  3. Download .RIS

Abstract

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex.

Article and author information

Author details

  1. Ruming Chen

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  2. Cláudia Rato

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  3. Yahui Yan

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  4. Ana Crespillo-Casado

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  5. Hanna J Clarke

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  6. Heather P Harding

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  7. Stefan J Marciniak

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  8. Randy J Read

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.

  9. David Ron

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    dr360@medschl.cam.ac.uk
    Competing interests
    David Ron, Reviewing editor, eLife.

Copyright

© 2015, Chen 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

  • 3,408
    views
  • 774
    downloads
  • 72
    citations

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

Citations by DOI

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. Ruming Chen
  2. Cláudia Rato
  3. Yahui Yan
  4. Ana Crespillo-Casado
  5. Hanna J Clarke
  6. Heather P Harding
  7. Stefan J Marciniak
  8. Randy J Read
  9. David Ron
(2015)
G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases
eLife 4:e04871.
https://doi.org/10.7554/eLife.04871

Share this article

https://doi.org/10.7554/eLife.04871

Categories and tags

Research organisms

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

    Ana Crespillo-Casado, Joseph E Chambers ... David Ron
    Research Article Updated

    Dephosphorylation of translation initiation factor 2 (eIF2α) terminates signalling in the mammalian integrated stress response (ISR) and has emerged as a promising target for modifying the course of protein misfolding diseases. The [(o-chlorobenzylidene)amino]guanidines (Guanabenz and Sephin1) have been proposed to exert protective effects against misfolding by interfering with eIF2α-P dephosphorylation through selective disruption of a PP1-PPP1R15A holophosphatase complex. Surprisingly, they proved inert in vitro affecting neither stability of the PP1-PPP1R15A complex nor substrate-specific dephosphorylation. Furthermore, eIF2α-P dephosphorylation, assessed by a kinase shut-off experiment, progressed normally in Sephin1-treated cells. Consistent with its role in defending proteostasis, Sephin1 attenuated the IRE1 branch of the endoplasmic reticulum unfolded protein response. However, repression was noted in both wildtype and Ppp1r15a deleted cells and in cells rendered ISR-deficient by CRISPR editing of the Eif2s1 locus to encode a non-phosphorylatable eIF2α (eIF2αS51A). These findings challenge the view that [(o-chlorobenzylidene)amino]guanidines restore proteostasis by interfering with eIF2α-P dephosphorylation.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    Joseph E Chambers, Lucy E Dalton ... Stefan J Marciniak
    Research Article Updated

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR.