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

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Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein–Protein Interactions
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2017-08-01 00:00:00 , DOI: 10.1021/acschembio.7b00361
Søren W. Pedersen ₁ , Louise Albertsen ₁ , Griffin E. Moran ₁ , Brié Levesque ₂ , Stine B. Pedersen ₁ , Lina Bartels ₁ , Hannah Wapenaar ₁ , Fei Ye _{3,

4} , Mingjie Zhang _{3,

4} , Mark E. Bowen ₂ , Kristian Strømgaard ₁

Affiliation

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.

中文翻译：

特定位置的PSD-95 PDZ域的磷酸化揭示了蛋白质-蛋白质相互作用的微调调控。

突触后密度蛋白95 kDa（PSD-95）是关键的支架蛋白，通过其PDZ域与受体，离子通道和酶的C末端的相互作用，控制大脑突触中的信号传导。PSD-95受磷酸化高度调节。为了探讨磷酸化对PSD-95的影响，我们使用了半合成策略在PDZ域内的四个位置引入了磷酸化的氨基酸，并研究了其对与大量结合配偶体相互作用的影响。我们观察到了对亲和力的复杂影响。最值得注意的是，如NMR和单分子FRET所证实，Y397处的磷酸化诱导了对stargazin亲和力的显着提高。此外，我们比较了磷酸化对拟磷酸酶突变的影响，这表明拟磷酸酶不能有效替代酪氨酸磷酸化。我们生成位点特异性磷酸化PDZ域的策略提供了对磷酸化在PSD-95相互作用调控中的作用的详细了解。

更新日期：2017-08-02

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