Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity.

神经递质释放（neurotransmitter release）由突触囊泡（synaptic vesicles）与突触前质膜（presynaptic plasma membrane）的快速钙触发融合所介导，随后伴随突触囊泡膜的内吞作用（endocytosis）与再循环。尽管调控上述过程的诸多蛋白质已被探明，但其调控机制的研究仍处于起步阶段。本研究应用定量磷酸化蛋白质组学（quantitative phosphoproteomics）技术，对从Wistar大鼠（Wistar rats）脑内分离的静息与激活神经末梢中突触前蛋白质（presynaptic proteins）的磷酸化水平变化进行了鉴定。通过严谨的定量分析，我们共鉴定出252个在钙依赖性胞吐作用（calcium-dependent exocytosis）触发后出现上调或下调的磷酸化位点。其中，突触前活性区（presynaptic active zone）的蛋白质组分（包括bassoon、piccolo以及RIM1）内的磷酸化位点调控变化尤为显著。此外，本研究还绘制了刺激后被活化的激酶（kinases）与磷酸酶（phosphatases）的作用图谱。总体而言，本研究为突触前活动相关的磷酸化修饰变化提供了全景式快照，并为参与突触可塑性（presynaptic plasticity）的关键磷酸化位点的后续功能分析奠定了坚实的研究基础。