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.
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.
A Bidirectional Switch in the Shank3 Phosphorylation State Is Necessary to Enable Synaptic Scaling Up and DownMassIVE, doi:10.25345/C5CG2H.
- Gina G Turrigiano
- Gina G Turrigiano
- Pierre MJ Beltran
- Jeffrey R Cottrell
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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).
- Lisa M Monteggia, Vanderbilt University, United States
© 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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