Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells
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.
Reviewing Editor
- 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
- Received: February 14, 2019
- Accepted: July 6, 2019
- Accepted Manuscript published: July 8, 2019 (version 1)
- 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.
Metrics
-
- 1,781
- views
-
- 218
- downloads
-
- 12
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While d-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal d-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a d-serine transporter previously uncharacterized in the kidney and discovered d-serine as a non-canonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal d-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.
-
- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.