Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells

  1. Ben F Brian
  2. Adrienne S Jolicoeur
  3. Candace R Guerrero
  4. Myra G Nunez
  5. Zoi E Sychev
  6. Siv A Hegre
  7. Pål Sætrom
  8. Nagy Habib
  9. Justin M Drake
  10. Kathryn L Schwertfeger
  11. Tanya S Freedman  Is a corresponding author
  1. University of Minnesota, United States
  2. Norwegian University of Science and Technology, Norway
  3. Imperial College London, United Kingdom

Abstract

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for graphs in Figure 1, Figure 1-figure supplement 1, Figure 2, Figure 3, Figure 3-figure supplement 2, Figure 4, Figure 4-figure supplement 1, Figure 4-figure supplement 5, Figure 5, Figure 6, Figure 6-figure supplement 1, Figure 7, Figure 8, and Figure 9.Data sets and calibration curves resulting from our targeted mass spectrometry studies have been deposited in Panorama Public (https://panoramaweb.org/project/Panorama%20Public/begin.view?)

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Ben F Brian

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Adrienne S Jolicoeur

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Candace R Guerrero

    College of Biological Sciences Center for Mass Spectrometry and Proteomics, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Myra G Nunez

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Zoi E Sychev

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Siv A Hegre

    Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
    Competing interests
    The authors declare that no competing interests exist.
  7. Pål Sætrom

    Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
    Competing interests
    The authors declare that no competing interests exist.
  8. Nagy Habib

    Department of Surgery and Cancer, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Justin M Drake

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Kathryn L Schwertfeger

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Tanya S Freedman

    Department of Pharmacology, University of Minnesota, Minneapolis, United States
    For correspondence
    tfreedma@umn.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5168-5829

Funding

NIH Office of the Director (R01AR073966)

  • Tanya S Freedman

NIH Office of the Director (R03AI130978)

  • Tanya S Freedman

American Cancer Society (UMN IRG-58-001-55)

  • Tanya S Freedman

University of Minnesota (Grant-in-Aid #92286)

  • Tanya S Freedman

University of Minnesota (Research and Equipment Award NF-0315-02)

  • Tanya S Freedman

University of Minnesota (Center for Autoimmune Diseases Research Pilot Grant)

  • Tanya S Freedman

NIH Office of the Director (R01CA215052)

  • Kathryn L Schwertfeger

NIH Office of the Director (T32DA007097)

  • Ben F Brian

Research Council of Norway (230338)

  • Pål Sætrom

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

Reviewing Editor

  1. Roger J Davis, University of Massachusetts Medical School, United States

Ethics

Animal experimentation: All animal use complies with University of Minnesota (UMN) and National Institutes of Health (NIH) policy (Animal Welfare Assurance Number A3456-01). UMN is accredited by AAALAC, and all animal use was approved by the UMN Institutional Animal Care and Use Committee (IACUC, protocol # 1603-33559A). Animals are kept under supervision of a licensed doctor of veterinary medicine and supporting veterinary staff under strict NIH guidelines.

Version history

  1. Received: February 14, 2019
  2. Accepted: July 6, 2019
  3. Accepted Manuscript published: July 8, 2019 (version 1)
  4. Version of Record published: July 26, 2019 (version 2)

Copyright

© 2019, Brian 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. Ben F Brian
  2. Adrienne S Jolicoeur
  3. Candace R Guerrero
  4. Myra G Nunez
  5. Zoi E Sychev
  6. Siv A Hegre
  7. Pål Sætrom
  8. Nagy Habib
  9. Justin M Drake
  10. Kathryn L Schwertfeger
  11. Tanya S Freedman
(2019)
Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells
eLife 8:e46043.
https://doi.org/10.7554/eLife.46043

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https://doi.org/10.7554/eLife.46043

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