Differences in Src phosphorylation of PSD-93 and PSD-95 drive differences in scaffolding activity

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Scaffold proteins like Membrane-Associated Guanylate Kinase (MAGuK) proteins, including PSD-93 and PSD-95, organize synaptic function by bringing together interacting proteins.
• These MAGuKs share conserved domains but exhibit opposing roles in synaptic plasticity, influenced by Src kinase-mediated tyrosine phosphorylation.
• The precise effects of Src kinase phosphorylation on MAGuK structure, client protein interactions, and scaffolding activity remain largely uncharacterized.

Purpose of the Study:

• To investigate the in vitro phosphorylation of PSD-93 and PSD-95 by Src kinase.
• To determine how phosphorylation affects the binding affinities of PSD-93 and PSD-95 for postsynaptic client proteins.
• To elucidate the impact of phosphorylation on MAGuK-mediated supercomplex formation, biomolecular condensation, and dynamics.

Main Methods:

• In vitro kinase assays to characterize PSD-93 and PSD-95 phosphorylation by Src kinase.
• Biochemical assays to measure changes in client protein binding affinity upon phosphorylation.
• Small-angle X-ray scattering (SAXS) and single-molecule Förster Resonance Energy Transfer (smFRET) to assess structural and dynamic alterations.
• Analysis of biomolecular condensation and supercomplex formation.

Main Results:

• PSD-93 and PSD-95 are multiply phosphorylated by Src kinase at similar sites, with PSD-93 being a more robust substrate.
• Phosphorylation differentially alters the affinity of PSD-93 and PSD-95 for postsynaptic client proteins, leading to opposing effects on client recruitment into supercomplexes.
• Phosphorylation influences biomolecular condensation in PSD-93 but not PSD-95, suggesting a dissociation between interaction affinity and phase separation.
• While supertertiary structure remains largely unchanged, phosphorylation alters protein dynamics, with PSD-95 functioning at low protein concentrations and PSD-93 at higher concentrations.

Conclusions:

• Src kinase phosphorylation differentially modulates the scaffolding activity and client interactions of PSD-93 and PSD-95.
• The distinct effects of phosphorylation on PSD-93 and PSD-95 suggest specialized roles in synapse maturation and plasticity, potentially related to their concentration-dependent behaviors.
• The findings highlight the complex regulation of synaptic function by scaffold protein phosphorylation and its impact on biomolecular condensation and protein dynamics.