title: 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 (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 pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plantPapaver rhoeaswere 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.
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Deborah J
authors.identifier: 0000-0002-6122-3377
authors.initials: DJ
authors.lastname: Eaves
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Tamanna
authors.identifier: 0000-0002-3979-8721
authors.initials: T
authors.lastname: Haque
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Richard L
authors.initials: RL
authors.lastname: Tudor
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Barron
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Cleidiane G
authors.identifier: 0000-0003-0934-0792
authors.initials: CG
authors.lastname: Zampronio
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Nicholas P J
authors.initials: NP
authors.lastname: Cotton
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Barend H J
authors.identifier: 0000-0003-0542-5510
authors.initials: BH
authors.lastname: de Graaf
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Scott A
authors.identifier: 0000-0002-0202-6154
authors.initials: SA
authors.lastname: White
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Helen J
authors.identifier: 0000-0003-4590-9384
authors.initials: HJ
authors.lastname: Cooper
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: F Christopher H
authors.identifier: 0000-0003-3507-722X
authors.initials: FC
authors.lastname: Franklin
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.).
authors.forename: Jeffery F
authors.initials: JF
authors.lastname: Harper
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT Birmingham, United Kingdom (D.J.E., T.H., R.L.T., C.G.Z., N.P.J.C., B.H.J.d.e.G., S.A.W., H.J.C., F.C.H.F., V.E.F.-T.); and v.e.franklin-tong@bham.ac.uk.|Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 (Y.B., J.F.H.) v.e.franklin-tong@bham.ac.uk.
authors.forename: Vernonica E
authors.identifier: 0000-0003-1782-8413
authors.initials: VE
authors.lastname: Franklin-Tong
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011058:Pollen / Q000201:enzymology* / Q000235:genetics; D011494:Protein Kinases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D007527:Isoenzymes; D016877:Oxidants; D010940:Plant Proteins; D006861:Hydrogen Peroxide; D011494:Protein Kinases; D043564:Inorganic Pyrophosphatase; D002118:Calcium