1. Biochemistry and Chemical Biology

The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling

  1. Nam Chu
  2. Thibault Viennet
  3. Hwan Bae
  4. Antonieta Salguero
  5. Andras Boeszoermenyi
  6. Haribabu Arthanari  Is a corresponding author
  7. Philip A Cole  Is a corresponding author
  1. Harvard Medical School, Brigham and Women's Hospital, United States
  2. Harvard Medical School, Dana-Farber Cancer Institute, United States
  3. Harvard Medical School, United States
https://doi.org/10.7554/eLife.59151
Share this article
Cite this article
  1. Nam Chu
  2. Thibault Viennet
  3. Hwan Bae
  4. Antonieta Salguero
  5. Andras Boeszoermenyi
  6. Haribabu Arthanari
  7. Philip A Cole
(2020)
The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling
eLife 9:e59151.
https://doi.org/10.7554/eLife.59151
  2. Download BibTeX
  3. Download .RIS

Abstract

Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.

Data availability

Source data: Enzyme kinetics, fluorescence binding data, western blots, SDSPAGE gels have been deposited in Dryad: http://dx.doi:10.5061/dryad.0p2ngf1xg

The following data sets were generated

Article and author information

Author details

  1. Nam Chu

    Medicine/BCMP, Harvard Medical School, Brigham and Women's Hospital, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9717-5007

  2. Thibault Viennet

    BCMP, Harvard Medical School, Dana-Farber Cancer Institute, Boston, United States
    Competing interests
    No competing interests declared.

  3. Hwan Bae

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5252-252X

  4. Antonieta Salguero

    Medicine/BCMP, Harvard Medical School, Brigham and Women's Hospital, Boston, United States
    Competing interests
    No competing interests declared.

  5. Andras Boeszoermenyi

    BCMP, Harvard Medical School, Dana-Farber Cancer Institute, Boston, United States
    Competing interests
    No competing interests declared.

  6. Haribabu Arthanari

    BCMP, Harvard Medical School, Dana-Farber Cancer Institute, Boston, United States
    For correspondence
    hari_arthanari@hms.harvard.edu
    Competing interests
    No competing interests declared.

  7. Philip A Cole

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    pacole@bwh.harvard.edu
    Competing interests
    Philip A Cole, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6873-7824

Funding

National Cancer Institute (CA74305)

  • Philip A Cole

Claudia Adams Barr Program in Innovative Cancer Research

  • Haribabu Arthanari

Austrian Science Fund (Schroedinger Fellowship)

  • Andras Boeszoermenyi

American Heart Association (19POST34380800)

  • Andras Boeszoermenyi

National Institutes of Health (EB002026)

  • Haribabu Arthanari

Kwanjeong Educational Foundation (pre-doctoral fellowship)

  • Hwan Bae

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

Reviewing Editor

  1. John Kuriyan, University of California, Berkeley, United States

Publication history

  1. Received: May 20, 2020
  2. Accepted: August 3, 2020
  3. Accepted Manuscript published: August 3, 2020 (version 1)
  4. Version of Record published: August 19, 2020 (version 2)

Copyright

© 2020, Chu 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

  • 4,584
    Page views
  • 508
    Downloads
  • 26
    Citations

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

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. Nam Chu
  2. Thibault Viennet
  3. Hwan Bae
  4. Antonieta Salguero
  5. Andras Boeszoermenyi
  6. Haribabu Arthanari
  7. Philip A Cole
(2020)
The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling
eLife 9:e59151.
https://doi.org/10.7554/eLife.59151

Categories and tags

Research organism

Further reading

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

    PH domain-mediated autoinhibition and oncogenic activation of Akt

    Hwan Bae, Thibault Viennet ... Philip A Cole
    Research Advance Updated

    Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt’s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu et al., 2020). Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

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

    Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk with altered NTD dynamics

    Sean M Braet, Theresa SC Buckley ... Ganesh S Anand
    Research Article Updated

    SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

    1. Biochemistry and Chemical Biology

    Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects

    Holly Y Chen, Manju Swaroop ... Anand Swaroop
    Research Article

    Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6,000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.