1. Biochemistry and Chemical Biology
  2. Immunology and Inflammation

Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen

  1. Neel H Shah
  2. Mark Löbel
  3. Arthur Weiss
  4. John Kuriyan  Is a corresponding author
  1. University of California, Berkeley, United States
  2. University of California, San Francisco, United States
https://doi.org/10.7554/eLife.35190
Share this article
Cite this article
  1. Neel H Shah
  2. Mark Löbel
  3. Arthur Weiss
  4. John Kuriyan
(2018)
Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen
eLife 7:e35190.
https://doi.org/10.7554/eLife.35190
  2. Download BibTeX
  3. Download .RIS

Abstract

The specificity of tyrosine kinases is predominantly attributed to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al. 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely-related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.

Data availability

The following previously published data sets were used

Article and author information

Author details

  1. Neel H Shah

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1186-0626

  2. Mark Löbel

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  3. Arthur Weiss

    Rosalind Russell/Ephraim P. Engleman Rheumatology Research Center, Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2414-9024

  4. John Kuriyan

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jkuriyan@mac.com
    Competing interests
    John Kuriyan, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4414-5477

Funding

Damon Runyon Cancer Research Foundation

  • Neel H Shah

National Institutes of Health (P01 AI091580)

  • Arthur Weiss

German Academic Exchange Service London

  • Mark Löbel

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

Copyright

© 2018, Shah 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

  • 5,878
    views
  • 738
    downloads
  • 56
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Citations by DOI

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

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)

  1. Neel H Shah
  2. Mark Löbel
  3. Arthur Weiss
  4. John Kuriyan
(2018)
Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen
eLife 7:e35190.
https://doi.org/10.7554/eLife.35190

Share this article

https://doi.org/10.7554/eLife.35190

Categories and tags

Research organism

Further reading

    1. Immunology and Inflammation
    2. Structural Biology and Molecular Biophysics

    An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

    Neel H Shah, Qi Wang ... John Kuriyan
    Research Article Updated

    The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.