Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity

Abstract

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 (PP i ) to inorganic phosphate P i , 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 Ca 2+ 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.

Vacuolar H+-Pyrophosphatase and Cytosolic Soluble Pyrophosphatases Cooperatively Regulate Pyrophosphate Levels in Arabidopsis thaliana

“…For PPa1 and PPa5, we detected two spots with different isoelectric point (pI) values (Supplemental Figure 2C). The lower pI spots might represent phosphorylated proteins, as the phosphorylation of plant sPPases has been reported (de Graaf et al, 2006;Eaves et al, 2017). The pI values of each spot were comparable to the calculated pI values of each isozyme.…”

Apple S‐RNase triggers inhibition of tRNA aminoacylation by interacting with a soluble inorganic pyrophosphatase in growing self‐pollen tubes in vitro

“…b). A recent study has also revealed that phosphorylation in poorly conserved N‐terminal regions significantly affects the catalytic activity of two family I sPPases, Pr‐p261a and Pr‐p261b, in poppy (Eaves et al ., ). This is consistent with our interaction results, implying that binding of the N‐terminal region may represent another way of modifying sPPase.…”

Self-incompatibility inPapaverpollen: programmed cell death in an acidic environment

“…Additionally, when combined with the presence of Ca 2+ and H2O2, the activities of the phosphomimic and phosphonull forms of both sPPases were further reduced at pH 7. The same activity tests using low pH levels related to SI (pH 6.8 to 5.5) demonstrated that, in addition to low pH, both Ca 2+ and H2O2 contribute to the inhibition of p26 activity ( Eaves et al ., 2017 ). Considering the rapid stimulation of Ca 2+ and ROS, as well as the later pH drop induced by SI, these observations suggest that SI not only triggers the Ca 2+ -dependent phosphorylation of Pr-p26 but also contributes to the reduction of sPPase activity by stimulating several intracellular events that cumulatively contribute to changes in intracellular conditions that inhibit sPPase activity.…”

“…Notably, sPPase activity assays using recombinant Pr-p26.1a and Pr-p26.1b proteins showed that the sPPase activity is pH dependent with activities being dramatically reduced in acidic conditions when compared to those at the normal physiological pH 7 ( Wilkins et al ., 2015 ). The phosphorylation sites for Pr-p26.1a and Pr-p26.1b have recently been mapped and, importantly, the sPPase activities of the p26 phosphomimic mutants were more sensitive to a low pH environment (6.8 to 5.5) compared to the wild-type enzymes or the corresponding phosphonull mutants ( Eaves et al ., 2017 ). Additionally, when combined with the presence of Ca 2+ and H2O2, the activities of the phosphomimic and phosphonull forms of both sPPases were further reduced at pH 7.…”