1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors

  1. Manoj K Rathinaswamy
  2. Zied Gaieb
  3. Kaelin D Fleming
  4. Chiara Borsari
  5. Noah J Harris
  6. Brandon J Moeller
  7. Matthias P Wymann
  8. Rommie E Amaro
  9. John E Burke  Is a corresponding author
  1. University of Victoria, Canada
  2. University of California, San Diego, United States
  3. University of Basel, Switzerland
Research Article
  • Cited 0
  • Views 884
  • Annotations
Cite this article as: eLife 2021;10:e64691 doi: 10.7554/eLife.64691

Abstract

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110g) playing key roles in immune signalling. p110g is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insights into regulatory mechanisms that control PI3Kg kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

Data availability

The crystallography data has been deposited in the protein data bank with accession numbers (PDB: 7JWE, 7JX0, 7JWZ). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository[83] with the dataset identifier PXD021132. All data generated or analyzed during this study are included in the manuscript and supporting files. Specifically biochemical kinase assay data are included in the source data files.

The following previously published data sets were used

Article and author information

Author details

  1. Manoj K Rathinaswamy

    Biochemistry and Microbiology, University of Victoria, Victoria, Canada
    Competing interests
    The authors declare that no competing interests exist.
  2. Zied Gaieb

    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Kaelin D Fleming

    Biochemistry and Microbiology, University of Victoria, Victoria, Canada
    Competing interests
    The authors declare that no competing interests exist.
  4. Chiara Borsari

    Department of Biomedicine, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4688-8362
  5. Noah J Harris

    Biochemistry and Microbiology, University of Victoria, Victoria, Canada
    Competing interests
    The authors declare that no competing interests exist.
  6. Brandon J Moeller

    Biochemistry and Microbiology, University of Victoria, Victoria, Canada
    Competing interests
    The authors declare that no competing interests exist.
  7. Matthias P Wymann

    Department of Biomedicine, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3349-4281
  8. Rommie E Amaro

    Chemistry and Biochemistry, National Biomedical Computation Resource, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. John E Burke

    Biochemistry and Microbiology, University of Victoria, Victoria, Canada
    For correspondence
    jeburke@uvic.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7904-9859

Funding

Cancer Research Society (CRS-24368)

  • John E Burke

Michael Smith Foundation for Health Research (17686)

  • John E Burke

Canadian Institutes of Health Research (New Investigator)

  • John E Burke

National Institutes of Health (GM132826)

  • Zied Gaieb
  • Rommie E Amaro

Stiftung FHNW (341)

  • Matthias P Wymann

Swiss National Science (310030_189065)

  • Matthias P Wymann

Novartis Foundation (14B095)

  • Matthias P Wymann

Innosuisse - Schweizerische Agentur für Innovationsförderung (37213.1 IP-LS)

  • Matthias P Wymann

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

Reviewing Editor

  1. Amy Andreotti, Iowa State University, United States

Publication history

  1. Received: November 8, 2020
  2. Accepted: March 3, 2021
  3. Accepted Manuscript published: March 4, 2021 (version 1)
  4. Version of Record published: March 12, 2021 (version 2)

Copyright

© 2021, Rathinaswamy 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

  • 884
    Page views
  • 140
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    Renata O Pereira et al.
    Research Article Updated

    Adrenergic stimulation of brown adipocytes alters mitochondrial dynamics, including the mitochondrial fusion protein optic atrophy 1 (OPA1). However, direct mechanisms linking OPA1 to brown adipose tissue (BAT) physiology are incompletely understood. We utilized a mouse model of selective OPA1 deletion in BAT (OPA1 BAT KO) to investigate the role of OPA1 in thermogenesis. OPA1 is required for cold-induced activation of thermogenic genes in BAT. Unexpectedly, OPA1 deficiency induced fibroblast growth factor 21 (FGF21) as a BATokine in an activating transcription factor 4 (ATF4)-dependent manner. BAT-derived FGF21 mediates an adaptive response by inducing browning of white adipose tissue, increasing resting metabolic rates, and improving thermoregulation. However, mechanisms independent of FGF21, but dependent on ATF4 induction, promote resistance to diet-induced obesity in OPA1 BAT KO mice. These findings uncover a homeostatic mechanism of BAT-mediated metabolic protection governed in part by an ATF4-FGF21 axis, which is activated independently of BAT thermogenic function.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Haitao Zhang et al.
    Tools and Resources

    The human kinome comprises 538 kinases playing essential functions by catalyzing protein phosphorylation. Annotation of subcellular distribution of the kinome greatly facilitates investigation of normal and disease mechanisms. Here, we present Kinome Atlas (KA), an image-based map of the kinome annotated to 10 cellular compartments. 456 epitope-tagged kinases, representing 85% of the human kinome, were expressed in HeLa cells and imaged by immunofluorescent microscopy under a similar condition. KA revealed kinase family-enriched subcellular localizations, and discovered a collection of new kinase localizations at mitochondria, plasma membrane, extracellular space, and other structures. Furthermore, KA demonstrated the role of liquid-liquid phase separation in formation of kinase condensates. Identification of MOK as a mitochondrial kinase revealed its function in cristae dynamics, respiration, and oxidative stress response. Although limited by possible mislocalization due to overexpression or epitope tagging, this subcellular map of the kinome can be used to refine regulatory mechanisms involving protein phosphorylation.