Publications

• Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.

  Draskovic P., Saiardi A., Bhandari R., Burton A., Ilc G., Kovacevic M., Snyder S.H., Podobnik M.

  Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is ab ... >> More

  Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo. << Less

  Chem Biol 15:274-286(2008) [PubMed] [EuropePMC]

• Structural and catalytic analyses of the InsP6 kinase activities of higher plant ITPKs.

  Zong G., Shears S.B., Wang H.

  Inositol phosphate signaling in plants is of substantial agricultural interest, with a considerable focus on the inositol tris/tetrakisphosphate kinase (ITPK) family of inositol phosphate kinases. Historically, the 4-6 isoforms of ITPKs that higher plants each express have been studied for their m ... >> More

  Inositol phosphate signaling in plants is of substantial agricultural interest, with a considerable focus on the inositol tris/tetrakisphosphate kinase (ITPK) family of inositol phosphate kinases. Historically, the 4-6 isoforms of ITPKs that higher plants each express have been studied for their multiplexing a metabolic pathway to synthesize inositol hexakisphosphate (ie InsP₆ or phytate), through the phosphorylation and dephosphorylation of multiple inositol phosphates, including Ins(1,3,4,5,6)P₅ (inositol-1,3,4,5,6-pentakisphosphate). A more recent discovery is ITPK-catalyzed phosphorylation of InsP₆ to inositol pyrophosphates, which regulate plant immunity and phosphate homeostasis. However, a molecular-based explanation for these alternate catalytic activities has been missing, because no plant ITPK structure has previously been solved. Herein, we provide biochemical and structural analyses of ITPKs from Zea mays and Glycine max. For this work we introduce a simple, enzyme-coupled microplate-based assay of InsP₆  kinase activity that should promote more general access to this important field. Furthermore, a ZmITPK1/InsP₆ crystal complex is described at a resolution of 2.6 Å, which identifies a number of catalytically important residues; their functionality is confirmed by mutagenesis. We further demonstrate that ZmITPK1 adds a β-phosphate to the 3-position of Ins(1,2,3,4,5)P₅ , yielding a candidate signal for regulating phosphate homeostasis. An impactful discovery is our description of a 29-residue catalytic specificity element; by interchanging this element between GmITPK1 and GmITPK2, we demonstrate how its isoform-specific sequence specifically determines whether the host protein phosphorylates InsP₆ , without substantially affecting Ins(1,3,4,5,6)P₅  metabolism. Our structural rationalization of key catalytic differences between alternate ITPK isoforms will complement future research into their functional diversity. << Less

  FASEB J. 36:e22380-e22380(2022) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress.

  Choi J.H., Williams J., Cho J., Falck J.R., Shears S.B.

  Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was foun ... >> More

  Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 microM). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/-0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes. << Less

  J. Biol. Chem. 282:30763-30775(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain.

  Gokhale N.A., Zaremba A., Shears S.B.

  The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a p ... >> More

  The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 K(d)=96 nM; PPIP5K2 K(d)=705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells. << Less

  Biochem. J. 434:415-426(2011) [PubMed] [EuropePMC]

• Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.

  Fridy P.C., Otto J.C., Dollins D.E., York J.D.

  Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved ... >> More

  Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events. << Less

  J. Biol. Chem. 282:30754-30762(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification and characterization of a novel inositol hexakisphosphate kinase.

  Saiardi A., Nagata E., Luo H.R., Snowman A.M., Snyder S.H.

  The inositol pyrophosphate disphosphoinositol pentakisphosphate (PP-InsP(3)/InsP(7)) is formed in mammals by two recently cloned inositol hexakiphosphate kinases, InsP(6)K1 and InsP(6)K2 (Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1 ... >> More

  The inositol pyrophosphate disphosphoinositol pentakisphosphate (PP-InsP(3)/InsP(7)) is formed in mammals by two recently cloned inositol hexakiphosphate kinases, InsP(6)K1 and InsP(6)K2 (Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326). We now report the identification, cloning, and characterization of a third InsP(7) forming enzyme designated InsP(6)K3. InsP(6)K3 displays 50 and 45% sequence identity to InsP(6)K1 and InsP(6)K2, respectively, with a smaller mass (46 kDa) and a more basic character than the other two enzymes. InsP(6)K3 is most enriched in the brain where its localization resembles InsP(6)K1 and InsP(6)K2. Intracellular disposition discriminates the three enzymes with InsP(6)K2 being exclusively nuclear, InsP(6)K3 predominating in the cytoplasm, and InsP(6)K1 displaying comparable nuclear and cytosolic densities. << Less

  J. Biol. Chem. 276:39179-39185(2001) [PubMed] [EuropePMC]

• A conserved family of enzymes that phosphorylate inositol hexakisphosphate.

  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.

  Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related ... >> More

  Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6. << Less

  Science 316:106-109(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.