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The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis*

  • Chunfang Gu
    Affiliations
    Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, 27709
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  • Hoai-Nghia Nguyen
    Affiliations
    Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, 27709
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  • Alexandre Hofer
    Affiliations
    Department of Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
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  • Henning J. Jessen
    Footnotes
    Affiliations
    Institute of Organic Chemistry, Albert Ludwigs University, Albertstrasse 21, 79104 Freiburg, Germany
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  • Xuming Dai
    Footnotes
    Affiliations
    Division of Cardiology, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina 27599
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  • Huanchen Wang
    Affiliations
    Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, 27709
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  • Stephen B. Shears
    Correspondence
    To whom correspondence should be addressed: Laboratory of Signal Transduction, NIEHS, National Institutes of Health, 111 T. W. Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-0793;
    Affiliations
    Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, 27709
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  • Author Footnotes
    * This work was supported in part by the Intramural Research Program of the NIEHS, National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Supported by Swiss National Science Foundation Grant PP00P2_157607.
    2 Partially supported by National Center for Advancing Translational Sciences, National Institutes of Health Grant UL1TR001111.
Open AccessPublished:January 26, 2017DOI:https://doi.org/10.1074/jbc.M116.765743
      Proteins responsible for Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [Pi] is “sensed” by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) as follows: during a period of Pi starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP7 levels to fall in parallel (Azevedo, C., and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219–231. Hitherto, such Pi sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP7 and ATP decrease upon [Pi] starvation and subsequently recover during Pi replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8), reacts more dramatically (i.e. with a wider dynamic range and greater sensitivity). To understand this novel InsP8 response, we characterized kinetic properties of the bifunctional 5-InsP7 kinase/InsP8 phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases (PPIP5Ks). These data fulfil previously published criteria for any bifunctional kinase/phosphatase to exhibit concentration robustness, permitting levels of the kinase product (InsP8 in this case) to fluctuate independently of varying precursor (i.e. 5-InsP7) pool size. Moreover, we report that InsP8 phosphatase activities of PPIP5Ks are strongly inhibited by Pi (40–90% within the 0–1 mm range). For PPIP5K2, Pi sensing by InsP8 is amplified by a 2-fold activation of 5-InsP7 kinase activity by Pi within the 0–5 mm range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate signaling, regulating InsP8 turnover independently of 5-InsP7, in response to fluctuations in extracellular supply of a key nutrient.

      Introduction

      Phosphate has multiple functions that direct the survival of all living organisms: in its organic form, Pi is a component of genomic material, it serves as an energy currency, and it is ubiquitous in cell signaling. Thus, Pi homeostasis is essential to life, but the mechanisms by which this occurs in humans and other metazoans are largely unknown (
      • Komaba H.
      • Fukagawa M.
      Phosphate-a poison for humans?.
      ,
      • Bergwitz C.
      • Jüppner H.
      Phosphate sensing.
      ). Most of the previous work in this field of research has focused on yeast models (
      • Lee Y.S.
      • Huang K.
      • Quiocho F.A.
      • O'Shea E.K.
      Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate.
      ,
      • Lenburg M.E.
      • O'Shea E.K.
      Signaling phosphate starvation.
      ,
      • Wild R.
      • Gerasimaite R.
      • Jung J.Y.
      • Truffault V.
      • Pavlovic I.
      • Schmidt A.
      • Saiardi A.
      • Jessen H.J.
      • Poirier Y.
      • Hothorn M.
      • Mayer A.
      Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.
      ). In particular, recent studies with Saccharomyces cerevisiae have revealed a new function in Pi homeostasis for inositol pyrophosphates (
      • Wild R.
      • Gerasimaite R.
      • Jung J.Y.
      • Truffault V.
      • Pavlovic I.
      • Schmidt A.
      • Saiardi A.
      • Jessen H.J.
      • Poirier Y.
      • Hothorn M.
      • Mayer A.
      Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.
      ). The latter are soluble, intracellular signals that contain multiple phosphates and diphosphates; up to seven (InsP7)
      The abbreviations used are: InsP7
      diphosphoinositol pentakisphosphate
      1-InsP7
      1-diphosphoinositol 2,3,4,5,6-pentakisphosphate
      InsP6
      inositol hexakisphosphate
      5-InsP7
      5-diphosphoinositol 1,2,3,4,6-pentakisphosphate
      InsP8
      1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate
      XPR1
      xenotropic and polytropic retrovirus receptor 1
      PPIP5K
      diphosphoinositol-pentakisphosphate kinase
      SPX
      SYG1/Pho81/XPR1 proteins
      IP6K
      inositol-hexakisphosphate kinase
      InsP5
      inositol pentakisphosphate
      DIPP
      diphosphoinositol-polyphosphate phosphohydrolase
      CRISPR
      clustered regularly interspaced short palindromic repeats.
      or eight (InsP8) phosphates in total are crammed around a six-carbon inositol ring (see Refs.
      • Saiardi A.
      How inositol pyrophosphates control cellular phosphate homeostasis?.
      ,
      • Shears S.B.
      Diphosphoinositol polyphosphates: metabolic messengers?.
      ,
      • Thota S.G.
      • Bhandari R.
      The emerging roles of inositol pyrophosphates in eukaryotic cell physiology.
      and Fig. 1). In S. cerevisiae, levels of one inositol pyrophosphate, 5-InsP7, track perturbations to Pi homeostasis (
      • Wild R.
      • Gerasimaite R.
      • Jung J.Y.
      • Truffault V.
      • Pavlovic I.
      • Schmidt A.
      • Saiardi A.
      • Jessen H.J.
      • Poirier Y.
      • Hothorn M.
      • Mayer A.
      Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.
      ).
      Figure thumbnail gr1
      FIGURE 1Inositol pyrophosphate metabolism. The schematic describes all known mammalian enzyme classes that interconvert InsP6 with InsP8. Note that current thinking (
      • Padmanabhan U.
      • Dollins D.E.
      • Fridy P.C.
      • York J.D.
      • Downes C.P.
      Characterization of a selective inhibitor of inositol hexakisphosphate kinases: use in defining biological roles and metabolic relationships of inositol pyrophosphates.
      ,
      • Weaver J.D.
      • Wang H.
      • Shears S.B.
      The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment.
      ) has the major route from InsP6 to InsP8 in mammalian cells progressing through 5-InsP7 rather than 1-InsP7.
      This Pi-sensing activity of 5-InsP7 appears to reflect it being synthesized by a kinase class (kcs1 in yeast; IP6Ks in metazoans) that exhibits an unusually low affinity for ATP (
      • Voglmaier S.M.
      • Bembenek M.E.
      • Kaplin A.I.
      • Dormán G.
      • Olszewski J.D.
      • Prestwich G.D.
      • Snyder S.H.
      Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.
      ,
      • Saiardi A.
      • Erdjument-Bromage H.
      • Snowman A.M.
      • Tempst P.
      • Snyder S.H.
      Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.
      ). Consequently, cellular levels of 5-InsP7 in yeast decrease in response to the drop in [ATP] that accompanies extracellular [Pi] depletion (
      • Wild R.
      • Gerasimaite R.
      • Jung J.Y.
      • Truffault V.
      • Pavlovic I.
      • Schmidt A.
      • Saiardi A.
      • Jessen H.J.
      • Poirier Y.
      • Hothorn M.
      • Mayer A.
      Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.
      ,
      • Lonetti A.
      • Szijgyarto Z.
      • Bosch D.
      • Loss O.
      • Azevedo C.
      • Saiardi A.
      Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases.
      ). Furthermore, these ATP-driven changes in 5-InsP7 levels appear to comprise a dynamic signaling response because 5-InsP7 regulates proteins that maintain Pi homeostasis through interactions with their SPX domains (
      • Wild R.
      • Gerasimaite R.
      • Jung J.Y.
      • Truffault V.
      • Pavlovic I.
      • Schmidt A.
      • Saiardi A.
      • Jessen H.J.
      • Poirier Y.
      • Hothorn M.
      • Mayer A.
      Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.
      ). However, it is not known to what extent this signaling response is applicable to metazoan cells, which lack orthologs of many of the yeast genes that function in Pi sensing and Pi homeostasis (
      • Bergwitz C.
      • Jüppner H.
      Phosphate sensing.
      ).
      In the current study, we have searched for links between Pi homeostasis and inositol pyrophosphates in a human model system: the HCT116 intestinal epithelial cell line. This choice reflects the physiological relevance of both small and large fluctuations in [Pi] within the gastrointestinal tract (
      • Marks J.
      • Lee G.J.
      • Nadaraja S.P.
      • Debnam E.S.
      • Unwin R.J.
      Experimental and regional variations in Na+-dependent and Na+-independent phosphate transport along the rat small intestine and colon.
      ). One of our goals has been to investigate whether there are changes in extracellular [Pi] that might cause intracellular [ATP] and [5-InsP7] to co-vary, which as mentioned above is considered to be an IP6K-dependent phenomenon.
      Perhaps as a consequence of 5-InsP7 being the most abundant of the inositol pyrophosphates, it has been the focus of much of the literature in this field (
      • Saiardi A.
      How inositol pyrophosphates control cellular phosphate homeostasis?.
      ,
      • Thota S.G.
      • Bhandari R.
      The emerging roles of inositol pyrophosphates in eukaryotic cell physiology.
      ,
      • Shears S.B.
      Inositol pyrophosphates: why so many phosphates?.
      ,
      • Saiardi A.
      Cell signalling by inositol pyrophosphates.
      ). In the current study, we also study a different inositol pyrophosphate, InsP8 (Fig. 1). We describe some new features to InsP8 turnover that solidify its own, independent cell signaling credentials. This information arises out of our focus on the PPIP5Ks (Fig. 2). The latter enzymes are of general interest; in addition to hosting a kinase domain that phosphorylates 5-InsP7 to InsP8, PPIP5Ks posses a separate phosphatase domain that dephosphorylates InsP8 back to 5-InsP7 (
      • Fridy P.C.
      • Otto J.C.
      • Dollins D.E.
      • York J.D.
      Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.
      ,
      • Mulugu S.
      • Bai W.
      • Fridy P.C.
      • Bastidas R.J.
      • Otto J.C.
      • Dollins D.E.
      • Haystead T.A.
      • Ribeiro A.A.
      • York J.D.
      A conserved family of enzymes that phosphorylate inositol hexakisphosphate.
      ,
      • Wang H.
      • Nair V.S.
      • Holland A.A.
      • Capolicchio S.
      • Jessen H.J.
      • Johnson M.K.
      • Shears S.B.
      Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S]2+ cluster which inhibits inositol pyrophosphate 1-phosphatase activity.
      ). That is, PPIP5Ks interconvert substrates and products in apparent “futile cycles” (FIGURE 1, FIGURE 2). Kinase/phosphatase and other covalent modification cycles are a nexus for regulatory inputs into metabolic and signaling pathways (
      • Newsholme E.A.
      • Arch J.R.
      • Brooks B.
      • Surholt B.
      The role of substrate cycles in metabolic regulation.
      ); in fact this phenomenon is considered a core motif in the field of systems biology (
      • Dasgupta T.
      • Croll D.H.
      • Owen J.A.
      • Vander Heiden M.G.
      • Locasale J.W.
      • Alon U.
      • Cantley L.C.
      • Gunawardena J.
      A fundamental trade-off in covalent switching and its circumvention by enzyme bifunctionality in glucose homeostasis.
      ). However, in general, such competing catalytic activities are hosted by separate proteins for the purposes of compartmentalization and for promoting signaling fidelity (
      • Levine J.
      • Kueh H.Y.
      • Mirny L.
      Intrinsic fluctuations, robustness, and tunability in signaling cycles.
      ). Only in rare cases have these apparent benefits been selected against in order that the mutually antagonistic catalytic activities co-exist within a single protein (
      • Dasgupta T.
      • Croll D.H.
      • Owen J.A.
      • Vander Heiden M.G.
      • Locasale J.W.
      • Alon U.
      • Cantley L.C.
      • Gunawardena J.
      A fundamental trade-off in covalent switching and its circumvention by enzyme bifunctionality in glucose homeostasis.
      ,
      • Straube R.
      Sensitivity and robustness in covalent modification cycles with a bifunctional converter enzyme.
      ,
      • Dexter J.P.
      • Dasgupta T.
      • Gunawardena J.
      Invariants reveal multiple forms of robustness in bifunctional enzyme systems.
      ). The PPIP5K family is one of these exceptions; representatives from humans, yeasts, and plants contain kinase and phosphatase domains (
      • Mulugu S.
      • Bai W.
      • Fridy P.C.
      • Bastidas R.J.
      • Otto J.C.
      • Dollins D.E.
      • Haystead T.A.
      • Ribeiro A.A.
      • York J.D.
      A conserved family of enzymes that phosphorylate inositol hexakisphosphate.
      ), indicating that this bifunctionality has survived at least 1.5 billion years of evolutionary pressure (
      • Hedges S.B.
      • Dudley J.
      • Kumar S.
      TimeTree: a public knowledge-base of divergence times among organisms.
      ).
      Figure thumbnail gr2
      FIGURE 2Domain graphic for human PPIP5Ks. Domain graphics are shown for the human PPIP5Ks used in this study (type 1, BC057395.1; type 2, XM_005271938). For PPIP5K1, amino acid residues defining each domain are numbered as in a previous study, which also defined the intrinsically disordered domain (IDR) (
      • Machkalyan G.
      • Trieu P.
      • Pétrin D.
      • Hébert T.E.
      • Miller G.J.
      PPIP5K1 interacts with the exocyst complex through a C-terminal intrinsically disordered domain and regulates cell motility.
      ). These boundaries were matched to those of the corresponding domains in PPIP5K2 by sequence alignments using Clustal Omega. The aligned intrinsically disordered domain boundaries in PPIP5K2 are consistent with those independently predicted from the PSIPRED Protein Sequence Analysis Workbench. The percent sequence identities across each of the three domains are also indicated. Also indicated are the nature and the locations of our engineered mutations in the kinase and phosphatase domains.
      Among a number of proposed advantages of having competing catalytic activities in a single, bifunctional protein are the following: (a) preventing signaling incoherence that can otherwise arise due to stochastic fluctuations in the degrees of expression of two separate proteins; (b) robustness, i.e. invariance to quantitative changes of the system's components, including substrate concentration; and (c) increased “parametric sensitivity,” that is, a situation in which signaling output is amplified following relatively small changes in the concentration of a particular parameter, such as an enzyme regulator (
      • Dasgupta T.
      • Croll D.H.
      • Owen J.A.
      • Vander Heiden M.G.
      • Locasale J.W.
      • Alon U.
      • Cantley L.C.
      • Gunawardena J.
      A fundamental trade-off in covalent switching and its circumvention by enzyme bifunctionality in glucose homeostasis.
      ,
      • Straube R.
      Sensitivity and robustness in covalent modification cycles with a bifunctional converter enzyme.
      ). However, the significance of these phenomena in vivo is dictated by the catalytic parameters of the mutually antagonistic domains (
      • Dasgupta T.
      • Croll D.H.
      • Owen J.A.
      • Vander Heiden M.G.
      • Locasale J.W.
      • Alon U.
      • Cantley L.C.
      • Gunawardena J.
      A fundamental trade-off in covalent switching and its circumvention by enzyme bifunctionality in glucose homeostasis.
      ,
      • Straube R.
      Sensitivity and robustness in covalent modification cycles with a bifunctional converter enzyme.
      ). Hitherto, we lacked this information. The full kinetic profile for PPIP5Ks has not previously been determined in the full-length versions of these enzymes. Moreover, there is no information in the literature describing the existence of a modulator of either the kinase or the phosphatase activity of any mammalian PPIP5K. The current study addresses these important gaps in our understanding of inositol pyrophosphate turnover. We demonstrate that Pi regulates the catalytic activities of the PPIP5Ks. Furthermore, our data indicate that InsP8 and 5-InsP7 each act through separate mechanisms to individually sense extracellular Pi status.

      Author Contributions

      C. G. performed most of the experiments and analyzed the results. H.-N. N. and H. W. also performed experiments and analyzed results. A. H. and H. J. J. synthesized essential reagents. C. G., H.-N. N., H. W., X. D., and S. B. S. contributed to project conception and the design of experiments. S. B. S. wrote most of the paper with contributions from all of the other coauthors.

      Acknowledgments

      We thank Dr. Jeremy Gunawardena for comments on a draft of the manuscript.

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