A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up or down scaling

Abstract

Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Using deepscale quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found wide-spread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the ASD-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were persistently hypo-phosphorylated during scaling up and transiently hyper-phosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. These mutations did not impact baseline synaptic strength, indicating that they gate, rather than drive, the induction of synaptic scaling. Thus an activity-dependent switch between hypo- and hyperphosphorylation at S1586 and S1615 of Shank3 enables scaling up or down, respectively. Collectively our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds, and can bias synapses toward upward or downward homeostatic plasticity.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data for Figures 2-7 have been provided.The original mass spectra and the protein sequence databases used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) and are accessible at ftp://MSV000087926@massive.ucsd.edu.

The following data sets were generated

Article and author information

Author details

  1. Chi-Hong Wu

    Department of Biology, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6391-0747
  2. Vedakumar Tatavarty

    Department of Biology, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Pierre MJ Beltran

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Andrea Guerrero

    Department of Biology, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5324-8232
  5. Hasmik Keshishian

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Karsten Krug

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Melanie A MacMullan

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5296-5902
  8. Li Li

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Steven A Carr

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Jeffrey R Cottrell

    Broad Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Gina G Turrigiano

    Department of Biology, Brandeis University, Waltham, United States
    For correspondence
    turrigiano@brandeis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4476-4059

Funding

National Institute of Neurological Disorders and Stroke (R35 NS111562)

  • Gina G Turrigiano

Simons Foundation Autism Research Initiative (345485)

  • Gina G Turrigiano

National Heart, Lung, and Blood Institute (F32 HL154711)

  • Pierre MJ Beltran

Stanley Center for Psychiatric Research, Broad Institute

  • Jeffrey R Cottrell

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

Ethics

Animal experimentation: All experimental procedures were performed according to NIH guidelines and were approved by the Broad Institute of MIT and Harvard IACUC (mouse cultures) or the Brandeis University IACUC (rat cultures, protocol # 21002).

Reviewing Editor

  1. Lisa M Monteggia, Vanderbilt University, United States

Publication history

  1. Received: September 28, 2021
  2. Preprint posted: October 3, 2021 (view preprint)
  3. Accepted: April 25, 2022
  4. Accepted Manuscript published: April 26, 2022 (version 1)
  5. Version of Record published: May 9, 2022 (version 2)

Copyright

© 2022, Wu 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.

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  1. Chi-Hong Wu
  2. Vedakumar Tatavarty
  3. Pierre MJ Beltran
  4. Andrea Guerrero
  5. Hasmik Keshishian
  6. Karsten Krug
  7. Melanie A MacMullan
  8. Li Li
  9. Steven A Carr
  10. Jeffrey R Cottrell
  11. Gina G Turrigiano
(2022)
A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up or down scaling
eLife 11:e74277.
https://doi.org/10.7554/eLife.74277
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