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 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 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.

Figure 1.

Phosphorylation sites identified by LC-MS/MS in recombinant p26 phosphorylated in vitro by endogenous pollen kinases. The sequence alignment of the N-terminal regions of the p26 sPPase proteins is annotated to indicate amino acid residues identified as phosphorylated by endogenous pollen kinases by LC-MS/MS. A to F show ETD mass spectra detected from p26a (A–C) and p26b (D–F) after phosphorylation using pollen extracts. A, Ser-13 phosphorylation recorded on +2 ions at mass-to-charge ratio (m/z) 809.85. B, Thr-18 phosphorylation recorded on +3 ions at m/z 399.19. C, Ser-27 phosphorylation detected on +2 ions at m/z 428.22. D, Thr-25 phosphorylation detected on +2 ions at m/z 491.25. E, Ser-41 phosphorylation detected on +3 ions at m/z 575.92. F, Ser-51 phosphorylation detected on +2 ions at m/z 523.24. S/T, Phosphorylated residues.

Figure 2.

Phosphorylation of recombinant p26 by CPKs in vitro. A and B, Incorporation of ³²P into p26 after in vitro kinase assays using recombinant AtCPK34, CKII, or PKA with recombinant p26a (A) and p26b (B). Top gels are autoradiographs showing ³²P incorporation (total pmol), and bottom gels are western blots to show equal loading of protein. C and D, Incorporation of ³²P into p26a (C) and p26b (D) during in vitro phosphorylation using PrCPK17/34, PrCPK14, PrCPK6/26, or AtCPK34. Values are means ± se (n = 3).

Figure 3.

Phosphorylation sites identified by LC-MS/MS in p26 sPPase proteins phosphorylated by CPKs. The sequence alignment of the N-terminal regions of the p26 sPPase proteins is annotated to indicate phosphorylations by individual recombinant kinases and whole pollen extract. S/T, Phosphorylated residues.

Figure 4.

Effects of pH on PPase activities of p26a/b and their phosphonull and phosphomimic mutant versions. Recombinant p26 and mutant versions were assayed for PPase activity at variable pH. Values are means ± se (n = 4). A, p26a. B, p26b.

Figure 5.

Ca²⁺ and H₂O₂ additively affect PPase activities of p26a/b and differentially affect their phosphonull and phosphomimic mutants. Recombinant p26 enzymes were assayed for PPase activity at pH 7 and supplemented with CaCl₂ and/or H₂O₂. Values are means ± se (n = 3).

Figure 6.

pH further reduces PPase activity in the presence of Ca²⁺ and H₂O₂. Recombinant sPPase p26a (A) and p26b (B) enzymes were assayed for PPase activity (diamonds) and in the presence of CaCl₂ (squares), H₂O₂ (circles), and CaCl₂ + H₂O₂ (triangles) at several pH levels. The activity for each was reduced further by lowered pH. Values are means ± se (n = 5 for p26a and n = 3 for p26b).