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

TY - JOUR

T1 - Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions

AU - Pedersen, Søren W

AU - Albertsen, Louise

AU - Moran, Griffin E

AU - Levesque, Brié

AU - Pedersen, Stine B

AU - Bartels, Lina

AU - Wapenaar, Hannah

AU - Ye, Fei

AU - Zhang, Mingjie

AU - Bowen, Mark E

AU - Strømgaard, Kristian

PY - 2017/9/15

Y1 - 2017/9/15

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Calcium Channels

KW - Disks Large Homolog 4 Protein

KW - Humans

KW - Intracellular Signaling Peptides and Proteins

KW - Membrane Proteins

KW - Models, Molecular

KW - PDZ Domains

KW - Phosphorylation

KW - Protein Folding

KW - Protein Interaction Maps

KW - Protein Stability

KW - Journal Article

U2 - 10.1021/acschembio.7b00361

DO - 10.1021/acschembio.7b00361

M3 - Journal article

C2 - 28692247

SN - 1554-8929

VL - 12

SP - 2313

EP - 2323

JO - A C S Chemical Biology

JF - A C S Chemical Biology

IS - 9

ER -