Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity
Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity
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DOI：
10.1104/pp.16.01450
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发表时间：
2017-03-01
期刊：
影响因子：
7.4
通讯作者：
Franklin-Tong, Vernonica E.
中科院分区：
文献类型：
--
作者：
Eaves, Deborah J.;Haque, Tamanna;Franklin-Tong, Vernonica E.
蛋白质磷酸化调节许多细胞过程。识别涉及的底物和蛋白激酶对于理解这些重要的翻译后修饰如何调节真核细胞的生物功能至关重要。焦磷酸酶催化无机磷酸盐(PPI)的水解为无机磷酸盐PI，推动生物合成反应；它们对于低细胞浓度的无机磷酸盐是必不可少的。最近有研究表明，I家族可溶性无机焦磷酸酶(SPPase)的翻译后调节可能影响其活性。我们先前已经证明，开花植物罂粟中的两个花粉表达的sPPase，Pr-p26.1a和Pr-p26.1b，被磷酸化抑制。尽管有潜在的意义，但关于sPPase的磷酸化和调控的数据很少。在这里，我们使用液相色谱串联质谱仪来定位这些花粉sPPase上原本不同的氨基末端延伸的磷酸化位点。尽管在文献中没有关于定位sPPase上的磷酸化位点的报道，但对各种蛋白质组的数据库调查发现了一些例子，表明磷酸化可能是调节这些酶的一种更广泛的机制。与未经修饰的蛋白质相比，Pr-p26.1a/b的拟磷突变体在pH 6.8时显著和不同地降低了PPase活性2.5倍，在钙和过氧化氢存在下降低了52%。这表明，关键部位的磷酸调节可以抑制这些蛋白质的催化反应，与关键的细胞内事件相一致。由于sPPase在真核细胞中的许多代谢途径中都是必不可少的，我们的发现发现sPPase的磷酸化是一种潜在的主要调节机制，可以用来减弱代谢。
Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollenexpressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca2+ and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.