Differences in Src phosphorylation of <scp>PSD</scp> ‐93 and <scp>PSD</scp> ‐95 drive differences in scaffolding activity

Scaffold proteins contain multiple binding modules that allow for co-localization of proteins that lack a direct interaction. Evolution resulted in different combinations of binding modules that rewired existing signal transduction pathways. The synaptic Membrane-Associated Guanylate Kinase (MAGuK) scaffold proteins post-synaptic density protein 93 (PSD-93) and post-synaptic density protein 95 (PSD-95) share the same organization of highly conserved binding domains connected by divergent linkers, which generates opposing functions in synaptic plasticity. Both are targets for tyrosine phosphorylation by Src kinase, but the extent and impacts of phosphorylation are poorly understood. Here, we characterized the in vitro phosphorylation of PSD-93 and PSD-95. Both were targeted at multiple sites that were similarly distributed, but PSD-93 was a more robust substrate. While Src kinase has been linked to synaptic plasticity, the direct effects on MAGuK function are not known. We found that phosphorylation differentially affected the affinity of PSD-93 and PSD-95 for postsynaptic client proteins. Phosphorylation also elicited opposite responses in recruitment of key synaptic clients by MAGuKs into supercomplexes, which we equate with scaffolding activity. Surprisingly, phosphorylation affected biomolecular condensation in PSD-93 but not PSD-95, which suggests that the affinity of protein interactions is disconnected from phase separation. Phosphorylation had minimal impacts on the supertertiary structure as assessed by small angle x-ray scattering although single molecule fluorescence resonance energy transfer revealed altered dynamics leaving the origin of functional effects unclear. PSD-95 appears optimized to work with small protein numbers while PSD-93 modulates condensation at higher protein concentrations. This difference suggests action at different stages of synapse maturation, which could be linked to synaptic plasticity.