Global Phosphoproteomic Analysis Reveals Diverse Functions of Serine/Threonine/Tyrosine Phosphorylation in the Model Cyanobacterium Synechococcus sp. Strain PCC 7002

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser, Thr, and Tyr residues. However, only a few dozen phosphorylation sites in cyanobacteria are known, presenting a major obstacle for further understanding the regulatory roles of reversible phosphorylation in this group of bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the model cyanobacterium Synechococcus sp. PCC 7002. In total, 280 phosphopeptides and 410 phosphorylation sites from 245 Synechococcus sp. PCC 7002 proteins were identified through the combined use of protein/peptide prefractionation, TiO2 enrichment, and LC–MS/MS analysis. The identified phosphoproteins were functionally categorized into an interaction map and found to be involved in various biological processes such as two-component signaling pathway and photosynthesis. Our data provide the first global survey of phosphorylation in cyanobacteria by using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided data set may help reveal the physiological functions underlying Ser/Thr/Tyr phosphorylation and facilitate the elucidation of the entire signaling networks in cyanobacteria.越来越多的证据表明，丝氨酸(serine, Ser)、苏氨酸(threonine, Thr)与酪氨酸(tyrosine, Tyr)残基上的蛋白质磷酸化是细菌中主要的翻译后修饰(post-translational modifications)类型之一，参与调控诸多生理过程。蓝细菌(Cyanobacteria)是最大的细菌类群之一，也是唯一具备产氧光合作用能力的原核生物。诸多蓝细菌菌株拥有数量异常庞大的、对Ser、Thr及Tyr残基具有特异性的蛋白激酶与蛋白磷酸酶。但目前已知的蓝细菌磷酸化位点仅数十个，这成为进一步解析可逆磷酸化在该类细菌中调控功能的主要阻碍。本研究针对模式蓝细菌聚球藻(Synechococcus sp.) PCC 7002开展了全局且位点特异性的磷酸化蛋白质组学(phosphoproteomic)分析。通过联合运用蛋白/肽段预分级、TiO₂富集与液相色谱-串联质谱(LC–MS/MS)分析手段，本研究从245个聚球藻PCC 7002蛋白中共鉴定得到280条磷酸肽与410个磷酸化位点。研究人员将鉴定到的磷酸化蛋白进行功能分类并构建相互作用图谱，发现其参与双组分信号传导通路(two-component signaling pathway)、光合作用等多种生物学过程。本研究数据首次通过磷酸化蛋白质组学方法实现了对蓝细菌磷酸化的全局调研，揭示了该修饰广泛的调控范畴。本数据集将有助于揭示Ser/Thr/Tyr磷酸化的生理功能，并推动蓝细菌完整信号网络的解析工作。