Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity

Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important post-translational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyse the hydrolysis of PPi to Pi, driving biosynthetic reactions; they are essential for low cytosolic PPi. It was recently suggested that post-translational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed 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 have used liquid chromatographic tandem mass spectrometry (LC-MS/MS) to map phosphorylation sites to the otherwise divergent N-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 H2O2 over unmodified proteins. This indicates that phospho-regulation 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.