Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells
- University of Minnesota, United States
- Norwegian University of Science and Technology, Norway
- 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?)
Article and author information
Author details
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
© 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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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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