Site-specific phosphorylation of PSD-95 dynamically regulates the postsynaptic density as observed by phase separation

Summary Postsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem, and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD.
Graphical abstract Genetic incorporation of Ser phosphorylations in PSD and the effect on the dynamics of PSD condensates.
Highlights • Genetically introduced site-specific pSer in single, tandem and full-length PSD-95 • The inserted pSer residues altered the PSD dynamics as shown by phase separation • Phosphorylation of Ser78 in PSD-95 inhibited phase separation with GluN2B and Stg • Phosphorylation of Ser116 in PSD-95 promoted phase separation with Stg only
Biochemistry; Biomolecules; Protein structure aspects; Biophysics; Biophysical Chemistry