1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

Research Article
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Cite this article as: eLife 2020;9:e61509 doi: 10.7554/eLife.61509

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.

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.

Reviewing Editor

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

Publication 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.

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