Data_Sheet_1_The Phosphatase PP2A Interacts With ArnA and ArnB to Regulate the Oligomeric State and the Stability of the ArnA/B Complex.PDF

In the crenarchaeon Sulfolobus acidocaldarius, the archaellum, a type-IV pilus like motility structure, is synthesized in response to nutrient starvation. Synthesis of components of the archaellum is controlled by the archaellum regulatory network (arn). Protein phosphorylation plays an important role in this regulatory network since the deletion of several genes encoding protein kinases and the phosphatase PP2A affected cell motility. Several proteins in the archaellum regulatory network can be phosphorylated, however, details of how phosphorylation levels of different components affect archaellum synthesis are still unknown. To identify proteins interacting with the S. acidocaldarius phosphatases PTP and PP2A, co-immunoprecipitation assays coupled to mass spectrometry analysis were performed. Thirty minutes after growth in nutrient starvation medium, especially a conserved putative ATP/GTP binding protein (Saci_1281), a universal stress protein (Saci_0887) and the archaellum regulators ArnA and ArnB were identified as highly abundant interaction proteins of PP2A. The interaction between ArnA, ArnB, and PP2A was further studied. Previous studies showed that the Forkhead-associated domain containing ArnA interacts with von Willebrand type A domain containing ArnB, and that both proteins could be phosphorylated by the kinase ArnC in vitro. The ArnA/B heterodimer was reconstituted from the purified proteins. In complex with ArnA, phosphorylation of ArnB by the ArnC kinase was strongly stimulated and resulted in formation of (ArnA/B)2 and higher oligomeric complexes, while association and dephosphorylation by PP2A resulted in dissociation of these ArnA/B complexes.

在泉古菌（crenarchaeon）嗜酸热硫化叶菌（Sulfolobus acidocaldarius）中，古菌鞭毛（archaellum）作为一种类似IV型菌毛（type-IV pilus）的运动结构，会在营养匮乏条件下合成。古菌鞭毛组分的合成受古菌鞭毛调控网络（archaellum regulatory network，arn）调控。蛋白质磷酸化在该调控网络中发挥关键作用，因为敲除多个编码蛋白激酶及磷酸酶PP2A的基因会损害细胞运动能力。古菌鞭毛调控网络中的多种蛋白均可发生磷酸化，但目前仍未明确不同组分的磷酸化水平如何调控古菌鞭毛的合成。为鉴定与嗜酸热硫化叶菌的磷酸酶PTP及PP2A相互作用的蛋白，本研究开展了结合质谱分析的免疫共沉淀（co-immunoprecipitation）实验。在营养匮乏培养基中培养30分钟后，研究人员鉴定出多个与PP2A互作的高丰度蛋白，其中尤为突出的是保守推定ATP/GTP结合蛋白（Saci_1281）、通用应激蛋白（Saci_0887）以及古菌鞭毛调控蛋白ArnA和ArnB。研究团队针对ArnA、ArnB与PP2A之间的相互作用开展了更深入的探究。既往研究表明，含有叉头相关结构域（Forkhead-associated domain）的ArnA可与含有血管性血友病因子A型结构域（von Willebrand type A domain）的ArnB相互结合，且两种蛋白均可在体外被激酶ArnC磷酸化。研究人员通过纯化蛋白重组得到了ArnA/B异二聚体。在与ArnA形成复合物的状态下，ArnC激酶对ArnB的磷酸化会被显著增强，进而促成(ArnA/B)₂及更高阶寡聚复合物的形成；而PP2A介导的结合与去磷酸化则会导致这些ArnA/B复合物发生解离。