Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein–Protein Interactions

Abstract

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Postsynaptic localization and regulation of AMPA receptors and Cav1.2 by β2 adrenergic receptor/PKA and Ca 2+ /CaMKII signaling

“…If 400 PSD‐95 homologues are even distributed throughout the spine, the concentration would be ~10 μM. Consistently, K d values for the higher affinity GluN2 subunits are ~1 μM, whereas those for TARPs are in the range of 3–12 μM (Lim et al, ; Dakoji et al, ; Hafner et al, ; Pedersen et al, ).…”

“…The ESDV sequence at the C‐termini of GluN2A and 2B subunits constitutes a nearly optimal binding motif for those PDZ domains with Q or E being desirable at the −3 position and D or E the −1 position in addition to the previously established requirement of S/T at the −2 and V the 0 position (Lim et al, ; Zhu et al, ). The C‐terminal TTPV sequence of γ 2/3/4/8 is much less optimal; peptides mimicking the last 11 residues of GluN2A and 2B, which contain the C‐terminal ESDV sequence, possess a K d of ~1 μM for PDZ2 of PSD‐95, whereas peptides mimicking the last 10 residues of γ 2 , which contain the C‐terminal TTPV sequence, have a K d of ~12 μM (Lim et al, ; Dakoji et al, ; Pedersen et al, ) although extending the length of the γ 2 peptide decreases the K d to 3.4 μM (Hafner et al, ). Thus, NMDARs will tie up available PSD‐93/PSD‐95/SAP102 before AMPARs have access.…”

Emerging Themes in PDZ Domain Signaling

“…Similarly, Ser phosphorylation was identified in NHERF-1 PDZ1 domain and shown to reduce the binding to both the b2-adrenergic receptor and CFTR (Voltz et al, 2007). A recent study reported a comprehensive analysis of phosphorylation of four different sites on the three PDZ domains of PSD-95 (Pedersen et al, 2017). These phosphorylation sites were identified in vivo, although the kinases responsible for modifying these sites have not been identified.…”

“…In contrast to the PBM phosphorylation, the study of PDZ phosphorylation sites and consequences is underdeveloped, possibly owing to the challenges of obtaining phosphorylated PDZ domains. Phosphomimetic mutations, such as Asp and Glu residues, could be used, but they often do not represent authentic phosphorylation effectively, especially in the case of pTyr (Pedersen et al, 2017). Therefore, in vitro phosphorylation by recombinant kinases (Zhang et al, 2011a) or the expressed protein ligationebased semisynthetic strategy (Pedersen et al, 2017) shows great promise for biochemical and structural characterization of these interactions.…”

“…Phosphomimetic mutations, such as Asp and Glu residues, could be used, but they often do not represent authentic phosphorylation effectively, especially in the case of pTyr (Pedersen et al, 2017). Therefore, in vitro phosphorylation by recombinant kinases (Zhang et al, 2011a) or the expressed protein ligationebased semisynthetic strategy (Pedersen et al, 2017) shows great promise for biochemical and structural characterization of these interactions. Although our knowledge of how phosphorylation regulates ligand binding is limited, design of a phosphoswitchable PDZ domain has been achieved, suggesting the possibility that novel PDZ domains with switchable ligand binding can be designed for novel purposes (Smith et al, 2013).…”