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 hypo-phosphorylated during scaling up and 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. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ 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.

Microglial TNFα orchestrates protein phosphorylation in the cortex during the sleep period and controls homeostatic sleep

“…The latter is a central kinase in postsynaptic plasticity whose activity is controlled by phosphorylation at Thr 286 (Bayer & Schulman, 2019), also shown to be modified by TNFα. Another target phosphosite is Ser 1539 on the postsynaptic scaffold Shank3, recently shown to control synaptic availability of the protein and thereby expression of homeostatic plasticity (preprint: Wu et al , 2021). Examples can also be drawn on the regulation of presynaptic proteins by specific phosphorylations, herein identified as microglial TNFα‐dependent during the light period (Fig 4 and Dataset EV2).…”

Microglial TNFα orchestrates brain phosphorylation during the sleep period and controls homeostatic sleep

“…The latter is a central kinase in postsynaptic plasticity whose activity is controlled by phosphorylation at Thr 286 ( 37 ), also shown to be modified by TNFα. Another target phosphosite is Ser 1539 on the postsynaptic scaffold Shank3, recently shown to control synaptic availability of the protein and thereby expression of homeostatic plasticity ( 82 ). Examples can also be drawn on regulation of presynaptic proteins by specific phosphorylations, herein identified as microglial TNFα-dependent during sleep (fig.4 and table S2).…”

Quantifying phosphorylation dynamics in primary neuronal cultures using LC-MS/MS

“…Another point of note is sample throughput: This protocol does not include isobaric tagging of (phospho)-peptides for sample multiplexing ( Jones et al., 2020 ; Navarrete-Perea et al., 2018 ; Wu et al., 2021 ; Zecha et al., 2019 ). However, for large sample cohorts and/or if instrument time is limited, it can become necessary to use chemical tags (e.g., TMT-tags) to reduce overall measurement time.…”