Publications

• Integration of inositol phosphate signaling pathways via human ITPK1.

  Chamberlain P.P., Qian X., Stiles A.R., Cho J., Jones D.H., Lesley S.A., Grabau E.A., Shears S.B., Spraggon G.

  Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted ... >> More

  Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role. << Less

  J. Biol. Chem. 282:28117-28125(2007) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase 4 (AtITPK4) is an outlier to a family of ATP-grasp fold proteins from Arabidopsis.

  Sweetman D., Stavridou I., Johnson S., Green P., Caddick S.E., Brearley C.A.

  The Arabidopsis genome encodes a family of inositol 1,3,4-trisphosphate 5/6-kinases which form a subgroup of a larger group of ATP-grasp fold proteins. An analysis of the inositol 1,3,4-trisphosphate 5/6-kinase family might, ultimately, be best rewarded by detailed comparison of related enzymes in ... >> More

  The Arabidopsis genome encodes a family of inositol 1,3,4-trisphosphate 5/6-kinases which form a subgroup of a larger group of ATP-grasp fold proteins. An analysis of the inositol 1,3,4-trisphosphate 5/6-kinase family might, ultimately, be best rewarded by detailed comparison of related enzymes in a single genome. The enzyme encoded by At2G43980, AtITPK4; is an outlier to its family. At2G43980 is expressed in male and female organs of young and mature flowers. AtITPK4 differs from other family members in that it does not display inositol 3,4,5,6-tetrakisphosphate 1-kinase activity; rather, it displays inositol 1,4,5,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate isomerase activity. << Less

  FEBS Lett. 581:4165-4171(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme.

  Wilson M.P., Majerus P.W.

  Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and ... >> More

  Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified. << Less

  J. Biol. Chem. 271:11904-11910(1996) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase.

  Wilson M.P., Majerus P.W.

  We have sequenced and recombinantly expressed as a fusion protein an expressed sequence tag clone (GB Z25963) from Arabidopsis thaliana that represents the plant homologue of human inositol 1,3,4 trisphosphate 5/6-kinase. The 1365 base pair clone has an open reading frame of 960 base pairs that pr ... >> More

  We have sequenced and recombinantly expressed as a fusion protein an expressed sequence tag clone (GB Z25963) from Arabidopsis thaliana that represents the plant homologue of human inositol 1,3,4 trisphosphate 5/6-kinase. The 1365 base pair clone has an open reading frame of 960 base pairs that predicts a protein product of 36.2 kDa, with a pI of 6.1. There is no polyadenylation signal or poly (A) tail, suggesting that additional 3' sequence remains to be identified. The amino acid sequence is 30% identical to the human protein. There are several short regions with particularly high degrees of identity between the human and Arabidopsis protein sequences, and these may be useful in identifying the active site of the enzyme. The expressed sequence tag was expressed as a fusion protein in Escherichia coli, with a carboxyl terminal deletion removing one region of high identity between the two proteins. The protein product of this construct was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. The Arabidopsis enzyme produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 1:3, in contrast with the human enzyme which gives a product ratio of 3:1. << Less

  Biochem. Biophys. Res. Commun. 232:678-681(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• An Entamoeba histolytica inositol 1,3,4-trisphosphate 5/6-kinase has a novel 3-kinase activity.

  Field J., Wilson M.P., Mai Z., Majerus P.W., Samuelson J.

  Mol. Biochem. Parasitol. 108:119-123(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-trisphosphate 5/6-kinase.

  Miller G.J., Wilson M.P., Majerus P.W., Hurley J.H.

  Inositol hexakisphosphate and other inositol high polyphosphates have diverse and critical roles in eukaryotic regulatory pathways. Inositol 1,3,4-trisphosphate 5/6-kinase catalyzes the rate-limiting step in inositol high polyphosphate synthesis in animals. This multifunctional enzyme also has ino ... >> More

  Inositol hexakisphosphate and other inositol high polyphosphates have diverse and critical roles in eukaryotic regulatory pathways. Inositol 1,3,4-trisphosphate 5/6-kinase catalyzes the rate-limiting step in inositol high polyphosphate synthesis in animals. This multifunctional enzyme also has inositol 3,4,5,6-tetrakisphosphate 1-kinase and other activities. The structure of an archetypal family member, from Entamoeba histolytica, has been determined to 1.2 A resolution in binary and ternary complexes with nucleotide, substrate, and product. The structure reveals an ATP-grasp fold. The inositol ring faces ATP edge-on such that the 5- and 6-hydroxyl groups are nearly equidistant from the ATP gamma-phosphate in catalytically productive phosphoacceptor positions and explains the unusual dual site specificity of this kinase. Inositol tris- and tetrakisphosphates interact via three phosphate binding subsites and one solvent-exposed site that could in principle be occupied by 18 different substrates, explaining the mechanisms for the multiple specificities and catalytic activities of this enzyme. << Less

  Mol. Cell 18:201-212(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The pathway for the production of inositol hexakisphosphate in human cells.

  Verbsky J.W., Chang S.C., Wilson M.P., Mochizuki Y., Majerus P.W.

  The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase o ... >> More

  The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway. << Less

  J Biol Chem 280:1911-1920(2005) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Regulation of Ins(3,4,5,6)P(4) signaling by a reversible kinase/phosphatase.

  Ho M.W.Y., Yang X., Carew M.A., Zhang T., Hua L., Kwon Y.-U., Chung S.-K., Adelt S., Vogel G., Riley A.M., Potter B.V.L., Shears S.B.

  Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of ... >> More

  Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases. << Less

  Curr. Biol. 12:477-482(2002) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.