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

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

  1. Roman O Fedoryshchak
  2. Magdalena Přechová
  3. Abbey Butler
  4. Rebecca Lee
  5. Nicola O'Reilly
  6. Helen R Flynn
  7. Ambrosius P Snijders
  8. Noreen Eder
  9. Sila Ultanir
  10. Stephane Mouilleron  Is a corresponding author
  11. Richard Treisman  Is a corresponding author
  1. Francis Crick Institute, United Kingdom
https://doi.org/10.7554/eLife.61509
Share this article
Cite this article
  1. Roman O Fedoryshchak
  2. Magdalena Přechová
  3. Abbey Butler
  4. Rebecca Lee
  5. Nicola O'Reilly
  6. Helen R Flynn
  7. Ambrosius P Snijders
  8. Noreen Eder
  9. Sila Ultanir
  10. Stephane Mouilleron
  11. Richard Treisman
(2020)
Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme
eLife 9:e61509.
https://doi.org/10.7554/eLife.61509
  2. Download BibTeX
  3. Download .RIS

Abstract

PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin aII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.

Data availability

Diffraction data have been deposited in PDB under the accession code 6ZEE, 6ZEF, 6ZEG, 6ZEH, 6ZEI, 6ZEJ.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifiers PXD019977 and PXD019882.All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Roman O Fedoryshchak

    Signalling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1865-8372

  2. Magdalena Přechová

    Signalling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4591-854X

  3. Abbey Butler

    Signalling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  4. Rebecca Lee

    Signalling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Nicola O'Reilly

    Peptide Chemistry Science Technology Platform, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  6. Helen R Flynn

    Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7002-9130

  7. Ambrosius P Snijders

    Proteomics Science Technology Platform, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  8. Noreen Eder

    Kinases and Brain Development Laboratory, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  9. Sila Ultanir

    Kinases and Brain Development Laboratory, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  10. Stephane Mouilleron

    Structural Biology Science Technology Platform, Francis Crick Institute, London, United Kingdom
    For correspondence
    stephane.mouilleron@crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

  11. Richard Treisman

    Signalling and Transcription Group, Francis Crick Institute, London, United Kingdom
    For correspondence
    Richard.Treisman@Crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9658-0067

Funding

H2020 European Research Council (268690)

  • Richard Treisman

Cancer Research UK (FC001-190)

  • Sila Ultanir
  • Richard Treisman

Medical Research Council (FC001-190)

  • Sila Ultanir
  • Richard Treisman

Medical Research Council (FC001-190)

  • Sila Ultanir
  • Richard Treisman

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

Reviewing Editor

  1. Tony Hunter, Salk Institute for Biological Studies, United States

Ethics

Animal experimentation: Animal experimentation complied with all ethical regulations and was carried out under the UK Home Office Project licence P7C307997 in the Crick Biological Research Facilities.

Version history

  1. Received: August 7, 2020
  2. Accepted: September 24, 2020
  3. Accepted Manuscript published: September 25, 2020 (version 1)
  4. Version of Record published: October 30, 2020 (version 2)

Copyright

© 2020, Fedoryshchak 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

  • 3,059
    views
  • 385
    downloads
  • 22
    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. Roman O Fedoryshchak
  2. Magdalena Přechová
  3. Abbey Butler
  4. Rebecca Lee
  5. Nicola O'Reilly
  6. Helen R Flynn
  7. Ambrosius P Snijders
  8. Noreen Eder
  9. Sila Ultanir
  10. Stephane Mouilleron
  11. Richard Treisman
(2020)
Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme
eLife 9:e61509.
https://doi.org/10.7554/eLife.61509

Share this article

https://doi.org/10.7554/eLife.61509

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    MEMO1 binds iron and modulates iron homeostasis in cancer cells

    Natalia Dolgova, Eva-Maria E Uhlemann ... Oleg Y Dmitriev
    Research Article Updated

    Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease

    Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus

    Natalia E Ketaren, Fred D Mast ... John D Aitchison
    Research Advance

    To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

    1. Biochemistry and Chemical Biology

    Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

    Benjamin R Duewell, Naomi E Wilson ... Scott D Hansen
    Research Article

    Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.