Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs

  1. Bert van de Kooij
  2. Pau Creixell
  3. Anne van Vlimmeren
  4. Brian Joughin
  5. Chad J Miller
  6. Nasir Haider
  7. Craig D Simpson
  8. Rune Linding
  9. Vuk Stambolic
  10. Benjamin E Turk
  11. Michael B Yaffe  Is a corresponding author
  1. Massachusetts Institute of Technology, United States
  2. Yale School of Medicine, United States
  3. University of Toronto, Canada
  4. University of Copenhagen, Denmark
  5. Yale University School of Medicine, United States

Abstract

Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the -3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling.

Data availability

The Position Specific Scoring Matrices (PSSMs) containing the quantitative phosphorylation site motif information for each human Nek kinase have been deposited to the database of Scansite 4.0 (http://scansite.mit.edu). They are publicly available to use in all of the features of Scansite, including but not limited to motif prediction on given substrates, or motif-based database searches, simply by selecting the Nek kinase of interest from the dropdown menu presented upon selection of a specific feature. Please see the Scansite tutorial (https://scansite4.mit.edu/4.0/#tutorial) for details. The raw PSSMs are available in source Data File 1.The data published in Supplementary File 1 was obtained from Phosphositeplus (www.phosphosite.org), and can be accessed directly by performing a substrate search from the Phosphositeplus home page for the Nek kinase of interest. The data presented in Figure 7C was obtained by downloading the complete phosphorylation_site_dataset from Phosphositeplus (https://www.phosphosite.org/staticDownloads), which was analyzed by a custom-built script to score each site for their match to each Nek kinase motif according to the Scansite scoring algorithm.

Article and author information

Author details

  1. Bert van de Kooij

    Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Pau Creixell

    Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Anne van Vlimmeren

    Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Brian Joughin

    Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1022-9450
  5. Chad J Miller

    Department of Pharmacology, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Nasir Haider

    Department of Medical Biophysics, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  7. Craig D Simpson

    Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  8. Rune Linding

    Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  9. Vuk Stambolic

    Department of Medical Biophysics, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8853-3239
  10. Benjamin E Turk

    Department of Pharmacology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Michael B Yaffe

    Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    myaffe@mit.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9547-3251

Funding

National Institutes of Health (R01-GM104047)

  • Benjamin E Turk
  • Michael B Yaffe

Ludwig Institute for Cancer Research

  • Bert van de Kooij

Dutch Cancer Society (BUIT 2015-7546)

  • Bert van de Kooij

The Charles and Marjorie Holloway Foundation

  • Michael B Yaffe

National Institutes of Health (R01-ES015339)

  • Michael B Yaffe

National Institutes of Health (R35-ES028374)

  • Michael B Yaffe

National Cancer Institute (P30-CA14051)

  • Michael B Yaffe

National Institute of Environmental Health Sciences (P30-ES002109)

  • Michael B Yaffe

National Cancer Institute (K99CA226396)

  • Pau Creixell

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

Reviewing Editor

  1. Margaret C Frame, University of Edinburgh, United Kingdom

Version history

  1. Received: December 21, 2018
  2. Accepted: May 13, 2019
  3. Accepted Manuscript published: May 24, 2019 (version 1)
  4. Version of Record published: June 14, 2019 (version 2)

Copyright

© 2019, van de Kooij 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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  1. Bert van de Kooij
  2. Pau Creixell
  3. Anne van Vlimmeren
  4. Brian Joughin
  5. Chad J Miller
  6. Nasir Haider
  7. Craig D Simpson
  8. Rune Linding
  9. Vuk Stambolic
  10. Benjamin E Turk
  11. Michael B Yaffe
(2019)
Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs
eLife 8:e44635.
https://doi.org/10.7554/eLife.44635

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https://doi.org/10.7554/eLife.44635

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