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

Abstract
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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

1. Freedman T
(2019) Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells
Panorama.

http://panoramaweb.org/Freedman_LynA.url

The following previously published data sets were used

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Author details

Ben F Brian

Department of Pharmacology, University of Minnesota, Minneapolis, United States

Competing interests
The authors declare that no competing interests exist.
Adrienne S Jolicoeur

Department of Pharmacology, University of Minnesota, Minneapolis, United States

Competing interests
The authors declare that no competing interests exist.
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.
Myra G Nunez

Department of Pharmacology, University of Minnesota, Minneapolis, United States

Competing interests
The authors declare that no competing interests exist.
Zoi E Sychev

Department of Pharmacology, University of Minnesota, Minneapolis, United States

Competing interests
The authors declare that no competing interests exist.
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.
Pål Sætrom

Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway

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The authors declare that no competing interests exist.
Nagy Habib

Department of Surgery and Cancer, Imperial College London, London, United Kingdom

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The authors declare that no competing interests exist.
Justin M Drake

Department of Pharmacology, University of Minnesota, Minneapolis, United States

Competing interests
The authors declare that no competing interests exist.
Kathryn L Schwertfeger

Department of Pharmacology, University of Minnesota, Minneapolis, United States

Competing interests
The authors declare that no competing interests exist.
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.

"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.

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.

Copyright

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.