Small molecule inhibition of Csk alters affinity recognition by T cells

  1. Boryana N Manz
  2. Ying Xim Tan
  3. Adam Courtney
  4. Florentine Rutaganira
  5. Ed Palmer
  6. Kevan M Shokat
  7. Arthur Weiss  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Institute of Molecular and Cell Biology, Singapore
  3. University of basel, Switzerland

Abstract

The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk (CskAS) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of CskAS during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of CskAS enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.

Article and author information

Author details

  1. Boryana N Manz

    Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Ying Xim Tan

    Division of Cancer Genetics and Therapeutics, Laboratory of Chromatin, Epigenetics and Differentiation, Institute of Molecular and Cell Biology, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  3. Adam Courtney

    Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Florentine Rutaganira

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ed Palmer

    Departments of Biomedicine and Nephrology, University Hospital Basel, University of basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Kevan M Shokat

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Arthur Weiss

    Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, United States
    For correspondence
    art.weiss@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#AN086836, AN107127) of the University of California, San Francisco.

Copyright

© 2015, Manz 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

  • 4,315
    views
  • 800
    downloads
  • 40
    citations

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

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. Boryana N Manz
  2. Ying Xim Tan
  3. Adam Courtney
  4. Florentine Rutaganira
  5. Ed Palmer
  6. Kevan M Shokat
  7. Arthur Weiss
(2015)
Small molecule inhibition of Csk alters affinity recognition by T cells
eLife 4:e08088.
https://doi.org/10.7554/eLife.08088

Share this article

https://doi.org/10.7554/eLife.08088

Further reading

    1. Cell Biology
    2. Genetics and Genomics
    Keva Li, Nicholas Tolman ... UK Biobank Eye and Vision Consortium
    Research Article

    A glaucoma polygenic risk score (PRS) can effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study of the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic-risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve = 0.579), they offered marginal prediction improvement in PRS-only-based models (p=0.004). We identified a metabolomic signature associated with resilience in the top glaucoma PRS decile, with elevated glycolysis-related metabolites—lactate (p=8.8E-12), pyruvate (p=1.9E-10), and citrate (p=0.02)—linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (Pinteraction = 0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratiohighest vs. lowest total resilience metabolite quartile=0.71, 95% Confidence Interval = 0.64–0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (p=0.002) and optic nerve damage (p<0.0003) in Lmx1bV265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.

    1. Cell Biology
    Affiong Ika Oqua, Kin Chao ... Alejandra Tomas
    Research Article

    G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.