Regulation of Ins(3,4,5,6)P4 Signaling by a Reversible Kinase/Phosphatase
2002; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês
10.1016/s0960-9822(02)00713-3
ISSN1879-0445
AutoresMelisa W.Y. Ho, Xiaonian Yang, Mark A. Carew, Tong Zhang, Len Hua, Yong‐Uk Kwon, Sung‐Kee Chung, Stephan Adelt, Günter Vogel, Andrew M. Riley, Barry V. L. Potter, Stephen B. Shears,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoRegulation of Cl− channel conductance by Ins(3,4,5,6)P4 provides receptor-dependent control over salt and fluid secretion [1Carew M.A. Yang X. Schultz C. Shears S.B. Ins(3,4,5,6)P4 inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell-line.J. Biol. Chem. 2000; 275: 26906-26913Abstract Full Text Full Text PDF PubMed Google Scholar], cell volume homeostasis [2Nilius B. Prenen J. Voets T. Eggermont J. Bruzik K.S. Shears S.B. Droogmans G. Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl− currents in macrovascular endothelial cells.Pflugers Arch. 1998; 435: 637-644Crossref PubMed Scopus (29) Google Scholar], and electrical excitability of neurones and smooth muscle [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar]. Ignorance of how Ins(3,4,5,6)P4 is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P4 synthesis by Ins(1,3,4,5,6)P5 1-phosphatase activity by an enzyme previously characterized [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar] as an Ins(3,4,5,6)P4 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P3 as not simply an alternative kinase substrate [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar, 5Wilson M.P. Majerus P.W. Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning, and expression of recombinant enzyme.J. Biol. Chem. 1996; 271: 11904-11910Crossref PubMed Scopus (72) Google Scholar], but also an activator of Ins(1,3,4,5,6)P5 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P4 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 [6Woscholski R. Parker P.J. Inositol phosphatases: constructive destruction of phosphoinositides and inositol phosphates.in: Cockroft S. Biology of Phosphoinositides. Oxford University Press, Oxford2000: 320-338Google Scholar]. Regulation of Cl− channel conductance by Ins(3,4,5,6)P4 provides receptor-dependent control over salt and fluid secretion [1Carew M.A. Yang X. Schultz C. Shears S.B. Ins(3,4,5,6)P4 inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell-line.J. Biol. Chem. 2000; 275: 26906-26913Abstract Full Text Full Text PDF PubMed Google Scholar], cell volume homeostasis [2Nilius B. Prenen J. Voets T. Eggermont J. Bruzik K.S. Shears S.B. Droogmans G. Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl− currents in macrovascular endothelial cells.Pflugers Arch. 1998; 435: 637-644Crossref PubMed Scopus (29) Google Scholar], and electrical excitability of neurones and smooth muscle [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar]. Ignorance of how Ins(3,4,5,6)P4 is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P4 synthesis by Ins(1,3,4,5,6)P5 1-phosphatase activity by an enzyme previously characterized [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar] as an Ins(3,4,5,6)P4 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P3 as not simply an alternative kinase substrate [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar, 5Wilson M.P. Majerus P.W. Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning, and expression of recombinant enzyme.J. Biol. Chem. 1996; 271: 11904-11910Crossref PubMed Scopus (72) Google Scholar], but also an activator of Ins(1,3,4,5,6)P5 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P4 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 [6Woscholski R. Parker P.J. Inositol phosphatases: constructive destruction of phosphoinositides and inositol phosphates.in: Cockroft S. Biology of Phosphoinositides. Oxford University Press, Oxford2000: 320-338Google Scholar]. Signaling entities are frequently controlled by quite delicate shifts in the dynamic balance of regulatory signals with competing impacts. Ion channels provide particularly impressive examples of the degree of signal amplification that can result; switching the conductance state of a single channel can influence the transmembrane movement of millions of ions per second [7Clapham D. How to lose your hippocampus by working on chloride channels.Neuron. 2001; 29: 1-6Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. Both stimulatory (Ca2+ and CaMKII) and inhibitory [Ins(3,4,5,6)P4] signals converge on the family of so-called "Ca2+-activated" Cl− channels [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar, 8Ho M.W.Y. Kaetzel M.A. Armstrong D.L. Shears S.B. Regulation of a human chloride channel: a paradigm for integrating input from calcium, CaMKII and Ins(3,4,5,6)P4.J. Biol. Chem. 2001; 276: 18673-18680Crossref PubMed Scopus (55) Google Scholar, 9Xie W. Kaetzel M.A. Bruzik K.S. Dedman J.R. Shears S.B. Nelson D.J. Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84 colonic epithelial cells.J. Biol. Chem. 1996; 271: 14092-14097Crossref PubMed Scopus (112) Google Scholar]. Thus, receptor-dependent changes in Ins(3,4,5,6)P4 levels [10Menniti F.S. Oliver K.G. Nogimori K. Obie J.F. Shears S.B. Putney Jr., J.W. Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol (1,3,4,5,6) pentakisphosphate breakdown to myo-inositol (3,4,5,6) tetrakisphosphate.J. Biol. Chem. 1990; 265: 11167-11176Abstract Full Text PDF PubMed Google Scholar] is a topic of general biological significance, in that it impacts upon regulation of salt and fluid secretion from epithelial cells [1Carew M.A. Yang X. Schultz C. Shears S.B. Ins(3,4,5,6)P4 inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell-line.J. Biol. Chem. 2000; 275: 26906-26913Abstract Full Text Full Text PDF PubMed Google Scholar], cell volume homeostasis [2Nilius B. Prenen J. Voets T. Eggermont J. Bruzik K.S. Shears S.B. Droogmans G. Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl− currents in macrovascular endothelial cells.Pflugers Arch. 1998; 435: 637-644Crossref PubMed Scopus (29) Google Scholar], and electrical excitability in neurones and smooth muscle [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar, 11Frings S. Reuter D. Kleene S.J. Neuronal Ca2+-activated Cl− channels—homing in on an elusive channel species.Prog. Neurobiol. 2000; 60: 247-289Crossref PubMed Scopus (198) Google Scholar]. Unfortunately, understanding of the cellular control of Ins(3,4,5,6)P4 signaling has been rudimentary, because the pathway of Ins(3,4,5,6)P4 synthesis has not previously been characterized. One possibility is that Ins(3,4,5,6)P4 originates by phosphorylation of an InsP3 precursor. We examined all potential InsP3 precursors of Ins(3,4,5,6)P4 by individually introducing them at 100 μM concentrations into human pancreatoma CFPAC-1 cells; the bioassay for Ins(3,4,5,6)P4 synthesis by any endogenous kinase was the sensitivity of calcium-activated Cl− channels to Ins(3,4,5,6)P4 (IC50 = 3 μM) [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar, 12Ho M.W.Y. Shears S.B. Bruzik K.S. Duszyk M. French A.S. Inositol 3,4,5,6-tetrakisphosphate specifically inhibits a receptor-mediated Ca2+-dependent Cl− current in CFPAC-1 cells.Am. J. Physiol. 1997; 272: C1160-C1168PubMed Google Scholar]. Control whole-cell Cl− current (54 ± 3 pA/pF, n = 17) was reduced to 24 ± 3 pA/pF (n = 7) by 10 μM Ins(3,4,5,6)P4. In contrast, Ins(4,5,6)P3 (56 ± 7 pA/pF, n = 10), Ins(3,4,6)P3 (68 ± 5 pA/pF, n = 12), Ins(3,4,5)P3 (61 ± 7 pA/pF, n = 9), and Ins(3,5,6)P3 (60 ± 6 pA/pF, n = 13) showed no inhibitory effect. These results indicate that no substantial InsP3 phosphorylation to Ins(3,4,5,6)P4 occurred, while also demonstrating that all four phosphates of Ins(3,4,5,6)P4 contribute to its exquisite specificity of action. We therefore turned to the alternate possibility that Ins(3,4,5,6)P4 synthesis might involve dephosphorylation of InsP5; the simplest, most direct route would be by direct 1-phosphatase attack on Ins(1,3,4,5,6)P5. While cells possess active 3-phosphatase activity against this Ins(1,3,4,5,6)P5[13Chi H. Yang X. Kingsley P.D. O'Keefe R.J. Puzas J.E. Rosier R.N. Shears S.B. Reynolds P.R. Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo.Mol. Cell. Biol. 2000; 20: 6496-6507Crossref PubMed Scopus (46) Google Scholar], a 1-phosphatase activity has never been observed, even in tissues taken from 3-phosphatase "knock-out" mice [13Chi H. Yang X. Kingsley P.D. O'Keefe R.J. Puzas J.E. Rosier R.N. Shears S.B. Reynolds P.R. Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo.Mol. Cell. Biol. 2000; 20: 6496-6507Crossref PubMed Scopus (46) Google Scholar]. Thus, we speculated that if a 1-phosphatase were to exist, we might only observe it in vitro under specialized assay conditions. Since, in principle, a phosphokinase may be reversible to some degree if the ADP/ATP ratio is sufficiently high, we incubated the enzyme with 5 mM ADP, whereupon [3H]Ins(1,3,4,5,6)P5 was dephosphorylated to an [3H]InsP4 peak coeluting upon HPLC with a standard of Ins(3,4,5,6)P4(Figure 1A). Ins(1,4,5,6)P4 would also elute at this point, but we eliminated this option, as the [3H]InsP4 that was formed was a 1-kinase substrate (data not shown), which Ins(1,4,5,6)P4 is not [14Caffrey J.J. Darden T. Wenk M.R. Shears S.B. Expanding coincident signaling by PTEN through its Inositol 1,3,4,5,6-Pentakisphosphate 3-phosphatase Activity.FEBS Lett. 2001; 499: 6-10Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Other potential products of InsP5 dephosphorylation were all excluded by their earlier elution positions in this HPLC system (Figures 1B and 1C). In addition, we discovered (Figure 1B) that the Ins(1,3,4)P3 6-kinase activity of this same enzyme [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar] is also reversible. That is, Ins(1,3,4,6)P4 was dephosphorylated to Ins(1,3,4)P3(Figure 1B). However, these experiments also yielded several unexpected results. First, an additional, later-eluting InsP3 accumulated following Ins(1,3,4,6)P4 dephosphorylation, which is presumably Ins(3,4,6)P3 [not Ins(1,4,6)P3, see below]. Second, the kinase yielded an identical pattern of InsP3 products upon dephosphorylation of [3H]Ins(1,3,4,5)P4(Figure 1C), despite the latter not possessing a 6-phosphate. Third, Ins(1,3,4,6)P4 and Ins(1,3,4,5)P4 were interconverted (Figures 1B and 1C). Such "phosphomutase" activity is unprecedented in the field of inositol phosphate metabolism. To explain kinase reversibility and its phosphomutase activity, we noted earlier studies [15Wilcox R.A. Safrany S.T. Lampe D. Mills S.J. Nahorski S.R. Potter B.V.L. Modification at C2 of myo-inositol 1,4,5-trisphosphate produces inositol trisphosphates and tetrakisphosphates with potent biological activities.Eur. J. Biochem. 1994; 223: 115-124Crossref PubMed Scopus (42) Google Scholar], which were more recently applied to an inositol phosphate kinase from yeast [16Ongusaha P.P. Hughes P.J. Davey J. Michell R.H. Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation.Biochem. J. 1998; 335: 671-679Crossref PubMed Scopus (56) Google Scholar], showing that some inositol phosphates may interact with binding sites of receptors and enzymes in more than one orientation and that an inositol phosphate may mimic another by presenting key recognition features to the site in certain binding orientations. We propose a reaction pathway for the reversible kinase/phosphatase based on the idea that inositol phosphates bind in three different orientations (shown as modes 1, 2, and 3 in Figure 2), including two modes for Ins(1,3,4)P3. This new model (Figure 2) predicts that Ins(3,4,6)P3 is the later-eluting InsP3 peak in Figures 1B and 1C and further provides a novel explanation for two previously puzzling observations. First, we recently showed but could not satisfactorily explain Ins(1,2,4)P3 phosphorylation by this kinase at the 5 position [17Adelt S. Plettenburg O. Dallmann G. Ritter F.P. Shears S.B. Altenbach H.-J. Vogel G. Regiospecific phosphohydrolases from Dictyostelium as tools for the chemoenzymatic synthesis of the enantiomers D-myo-Inositol 1,2,4-trisphosphate and D-myo-Inositol 2,3,6-trisphosphate: non-physiological, potential analogues of biologically active D-myo-Inositol 1,3,4-trisphosphate.Bioorg. Med. Chem. Lett. 2001; 11: 2705-2708Crossref PubMed Scopus (19) Google Scholar]. Now we propose that Ins(1,2,4)P3 is recognized as a mode 3 substrate. Second, the ability of the kinase to phosphorylate Ins(1,3,4)P3 at the 5 and 6 positions [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar, 5Wilson M.P. Majerus P.W. Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning, and expression of recombinant enzyme.J. Biol. Chem. 1996; 271: 11904-11910Crossref PubMed Scopus (72) Google Scholar, 18Abdullah M. Hughes P.J. Craxton A. Gigg R. Desai T. Marecek J.F. Prestwich G.D. Shears S.B. Purification and characterization of inositol 1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.J. Biol. Chem. 1992; 267: 22340-22345Abstract Full Text PDF PubMed Google Scholar] is rationalized as reflecting two Ins(1,3,4)P3 binding modes (Figure 2), rather than a 5,6-cyclic intermediate [5Wilson M.P. Majerus P.W. Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning, and expression of recombinant enzyme.J. Biol. Chem. 1996; 271: 11904-11910Crossref PubMed Scopus (72) Google Scholar]. Our model (Figure 2), which assumes a single active site, provides the simplest explanation for our data, but we do not exclude more complex scenarios in which inositol phosphates may bind to more than one site on the protein. Assuming that our kinase/phosphatase utilizes a single active site, there are two phosphate groups on the inositol ring that are common to all binding modes and may therefore be structural determinants for substrate recognition (colored red in Figure 2). The position that is reversibly phosphorylated/dephosphorylated presumably also defines substrate recognition (also colored red in Figure 2). This model is consistent with other data showing the enzyme does not phosphory-late [3H]Ins(3,4)P2 (data not shown), nor Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, and Ins(1,4,5,6)P4[14Caffrey J.J. Darden T. Wenk M.R. Shears S.B. Expanding coincident signaling by PTEN through its Inositol 1,3,4,5,6-Pentakisphosphate 3-phosphatase Activity.FEBS Lett. 2001; 499: 6-10Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Abdullah M. Hughes P.J. Craxton A. Gigg R. Desai T. Marecek J.F. Prestwich G.D. Shears S.B. Purification and characterization of inositol 1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.J. Biol. Chem. 1992; 267: 22340-22345Abstract Full Text PDF PubMed Google Scholar]. While the groups colored red in Figure 2 are likely necessary for substrate recognition, they cannot be sufficient, because Ins(1,4)P2 itself is not phosphorylated (data not shown). Therefore, other features of the inositol phosphate ligands (colored green in Figure 2) must contribute to recognition, and some of these additional interactions are, presumably, specific to certain binding modes. The prediction that Ins(3,4,6)P3 is a type 1 substrate (Figure 2) was verified (Figure 3) using an online mass detection HPLC technique [19Mayr G.W. A novel metal-dye detection sysytem permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.Biochem. J. 1988; 254: 585-591Crossref PubMed Scopus (141) Google Scholar, 20Adelt S. Plettenburg O. Stricker R. Reiser G. Altenbach H.-J. Vogel G. Enzyme-assisted total synthesis of the optical antipodes D-myo-inositol 3,4,5-trisphosphate and D-myo-inositol 1,5,6-trisphosphate: aspects of their structure-activity relationship to biologically active inositol phosphates.J. Med. Chem. 1999; 42: 1262-1273Crossref PubMed Scopus (39) Google Scholar]. Kinetic data indicate a Km of 0.2 μM, a value close to that for Ins(1,3,4)P3[4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar]. We detected an InsP4 product eluting in the position expected of Ins(1,3,4,6)P4(Figure 3). Ins(1,3,4,5)P4 was also produced (Figure 3), although we cannot accurately quantify the Ins(1,3,4,6)P4/Ins(1,3,4,5)P4 ratio; the signal strength of this HPLC technique is not proportional to the number of phosphate groups [19Mayr G.W. A novel metal-dye detection sysytem permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.Biochem. J. 1988; 254: 585-591Crossref PubMed Scopus (141) Google Scholar] and even varies considerably between isomers containing the same number of phosphates [20Adelt S. Plettenburg O. Stricker R. Reiser G. Altenbach H.-J. Vogel G. Enzyme-assisted total synthesis of the optical antipodes D-myo-inositol 3,4,5-trisphosphate and D-myo-inositol 1,5,6-trisphosphate: aspects of their structure-activity relationship to biologically active inositol phosphates.J. Med. Chem. 1999; 42: 1262-1273Crossref PubMed Scopus (39) Google Scholar]. Nevertheless, Ins(3,4,6)P3 is clearly metabolized to Ins(1,3,4,5)P4(Figure 3), consistent with the reaction pathway shown in Figure 2. This involves dephosphorylation of Ins(1,3,4,6)P4 to Ins(1,3,4)P3 even though ATP (and not ADP) was added to these assays (Figure 2, Figure 3). Equally, the phosphorylation of Ins(1,3,4)P3 to Ins(1,3,4,6)P4 occurs when ADP (and not ATP) was added to the assays (Figure 1, Figure 2). Thus, the ability of the enzyme to act as both a kinase and a phosphatase is not entirely dictated by the ATP/ADP ratio added to the incubation media. It is possible that InsP4 participates more directly in the phosphotransferase reactions. One way in which Ins(1,3,4)P3 could be converted into Ins(1,3,4,6)P4 when Ins(1,3,4,5)P4 is the original substrate (Figure 1C) would be for Ins(1,3,4,5)P4 to donate its 5-phosphate group to the enzyme, forming a phosphorylenzyme intermediate, which then could transfer the phosphate to the 6-hydroxyl of Ins(1,3,4)P3, albeit in a manner apparently dependent upon some adenine nucleotide being present. Furthermore, in incubations containing 5 μM Ins(1,3,4,5,6)P5 and 5 mM ADP, net accumulation of Ins(3,4,5,6)P4 was increased by up to 5-fold upon addition of 1–5 μM Ins(1,3,4)P3(Figure 4A). One explanation for this result is that the newly formed Ins(3,4,5,6)P4 can be rephosphorylated, but less effectively in the presence of Ins(1,3,4)P3, a competing kinase substrate [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar]. Indeed, in these experiments, there was net phosphorylation of Ins(1,3,4)P3, but in a manner dependent upon Ins(1,3,4,5,6)P5(Figure 4B), which presumably donates the phosphate group, via a phosphorylenzyme intermediate, to either Ins(1,3,4)P3 or Ins(3,4,5,6)P4. Whatever the explanation, our exploration of several unusual features of this enzyme have led us to uncover assay conditions that optimize Ins(3,4,5,6)P4 synthesis (Figure 4A). With Ins(1,3,4)P3 present, the Vmax for Ins(1,3,4,5,6)P5 dephosphorylation was 82 pmol/μg protein/min, ∼10-fold less than the Vmax for Ins(3,4,5,6)P4 phosphorylation by these same preparations of recombinant enzyme (data not shown and see [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar]), although the latter reaction was previously estimated to operate at only 5%–10% of its capacity in receptor-activated cells [21Tan Z. Bruzik K.S. Shears S.B. Properties of the inositol 3,4,5,6-tetrakisphosphate 1-kinase purified from rat liver. Regulation of enzyme activity by inositol 1,3,4-trisphosphate.J. Biol. Chem. 1997; 272: 2285-2290Crossref PubMed Scopus (25) Google Scholar].Figure 4Ins(1,3,4)P3 Activates Ins(1,3,4,5,6)P5 1-Phosphatase ActivityShow full captionPanel (A) shows the degree of 1-phosphatase activity toward 5 μM [3H]Ins(1,3,4,5,6)P5 in 15 min assays performed as described in the legend to Figure 1; reactions were supplemented with either 0, 1, or 5 μM Ins(1,3,4)P3 plus 0.06 μg enzyme (means ± SE, n = 5). Additional incubations were performed with 5 μM [3H]Ins(1,3,4)P3 (∼1200 d.p.m.) plus either no Ins(1,3,4,5,6)P5 (open circles) or 5 μM nonradiolabeled Ins(1,3,4,5,6)P5 (closed circles); these reactions were assayed by HPLC (Synchropak Q100 column), and the InsP4 region of the chromatogram is shown in Panel (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Panel (A) shows the degree of 1-phosphatase activity toward 5 μM [3H]Ins(1,3,4,5,6)P5 in 15 min assays performed as described in the legend to Figure 1; reactions were supplemented with either 0, 1, or 5 μM Ins(1,3,4)P3 plus 0.06 μg enzyme (means ± SE, n = 5). Additional incubations were performed with 5 μM [3H]Ins(1,3,4)P3 (∼1200 d.p.m.) plus either no Ins(1,3,4,5,6)P5 (open circles) or 5 μM nonradiolabeled Ins(1,3,4,5,6)P5 (closed circles); these reactions were assayed by HPLC (Synchropak Q100 column), and the InsP4 region of the chromatogram is shown in Panel (B). Can this InsP5 1-phosphatase operate in vivo? Human colonic epithelial T84 cells were stably transfected with FLAG-tagged enzyme (theoretical size, 46.9 kDa). Expression was verified using anti-FLAG antibodies (Figure 5A, 49 ± 0.3 kDa, n = 3). Despite the enzyme acting in vitro as both an Ins(3,4,5,6)P4 1-kinase and Ins(1,3,4,5,6)P5 1-phosphatase, the elevated levels of [3H]Ins(3,4,5,6)P4 in enzyme-transfected cells indicate that phosphatase activity can predominate, specifically upon receptor activation (Figure 5B). Levels of [3H]InsP5 were not significantly affected by transfection (Figure 5C). The enhancement of the receptor-initiated Ins(3,4,5,6)P4 response in enzyme-transfected cells was accompanied by a 40% reduction in Ca2+-activated Cl− secretion (Figure 5D) from an intact cell monolayer. This provides a unique validation of the signaling importance of Ins(3,4,5,6)P4 in a physiological context. As well as regulating salt and fluid secretion, these Ca2+-activated Cl− channels mediate cell volume homeostasis and electrical excitability in neurones and smooth muscle [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar, 11Frings S. Reuter D. Kleene S.J. Neuronal Ca2+-activated Cl− channels—homing in on an elusive channel species.Prog. Neurobiol. 2000; 60: 247-289Crossref PubMed Scopus (198) Google Scholar], which testifies to the wide-ranging biological impact of our observations. Reciprocal coordination of the opposing 1-kinase/1-phosphatase reactions, catalyzed by a single enzyme, offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple phosphatases and kinases [6Woscholski R. Parker P.J. Inositol phosphatases: constructive destruction of phosphoinositides and inositol phosphates.in: Cockroft S. Biology of Phosphoinositides. Oxford University Press, Oxford2000: 320-338Google Scholar]. Finally, this demonstration that the InsP5 1-phosphatase regulates secretion (Figure 5) may also be of therapeutic interest. Upregulation of InsP5 1-phosphatase in airway epithelia could inhibit the gob-5 chloride channel that drives mucus secretion, which, when hyperresponsive, contributes to the asthmatic condition [22Nakanishi A. Morita S. Iwashita H. Sagiya Y. Ashida Y. Shirafuji H. Fujisawa Y. Nishimura O. Fujino M. Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma.Proc. Natl. Acad. Sci. USA. 2001; 98: 5175-5180Crossref PubMed Scopus (277) Google Scholar]. Downregulation of InsP5 1-phosphatase could be used as a strategy for enhancing Cl− secretion in the therapy of cystic fibrosis [3Ho M.W.Y. Carew M.A. Yang X. Shears S.B. Regulation of chloride channel conductance by Ins(3,4,5,6)P4: A phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3.in: Frontiers in Molecular Biology: Biology of Phosphoinositides. S. Cockroft. Oxford University Press, Oxford2000: 298-319Google Scholar]. Inositol phosphate phosphatase activity was assayed in buffer containing 100 mM KCl, 20 mM HEPES (pH 7.2), 5 mM ADP, 6 mM MgSO4, 0.3 mg/ml bovine serum albumin. Assays were acid quenched, neutralized, and analyzed by HPLC using a Synchropak Q100 column [14Caffrey J.J. Darden T. Wenk M.R. Shears S.B. Expanding coincident signaling by PTEN through its Inositol 1,3,4,5,6-Pentakisphosphate 3-phosphatase Activity.FEBS Lett. 2001; 499: 6-10Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]; 1 ml fractions were collected for 70 min, followed by 0.5 ml fractions. Inositol phosphate kinase activity was assayed in buffer containing 100 mM KCl, 20 mM HEPES (pH 7.2), 5 mM ATP, 10 mM phosphocreatine, 6 mM MgSO4, 10 μg/ml creatine phosphokinase (Calbiochem), 0.3 mg/ml bovine serum albumin. Some assays were acid quenched and analyzed by HPLC using a Synchropak Q100 column. Other reactions were heat inactivated (95°C, 3 min) and analyzed by a metal dye detection, HPLC method [19Mayr G.W. A novel metal-dye detection sysytem permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.Biochem. J. 1988; 254: 585-591Crossref PubMed Scopus (141) Google Scholar, 20Adelt S. Plettenburg O. Stricker R. Reiser G. Altenbach H.-J. Vogel G. Enzyme-assisted total synthesis of the optical antipodes D-myo-inositol 3,4,5-trisphosphate and D-myo-inositol 1,5,6-trisphosphate: aspects of their structure-activity relationship to biologically active inositol phosphates.J. Med. Chem. 1999; 42: 1262-1273Crossref PubMed Scopus (39) Google Scholar]. T84 cells were cultured at 37°C (5% CO2:95% air) in Iscove's Modified Dulbecco's medium (Hyclone) supplemented with 5% (w/v) FBS (Hyclone, Logan, UT), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.8 mg/ml G418 selection agent. The gene previously denoted a Ins(3,4,5,6)P4 1-kinase [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar] was cloned into PCMV-Tag4 expression vector between the BamHI and XhoI sites after the stop codon was removed. The transfection was carried out in a 60 mm dish, using LipoTAXI Mammalian Transfection Kit from Stratagene (La Jolla, CA), according to the manufacturer's instructions. Control transfections were performed at the same time using PCMV-Tag4 vector alone. Data shown are from one pair of cell lines stably transfected with either enzyme or vector; similar data were obtained with two additional, independently and stably transfected pairs of cell lines. T84 cells were detached by incubation with 0.25% (w/v) trypsin/0.02% (w/v) EDTA for 5–6 min. After trypsin removal, 200 μl aliquots (1.5 × 106/ml) were seeded into the circular wells (area = 0.45 cm2) of permeable supports, made by gluing (Silastic® sealant, Dow Corning, Midland, MI) a Sylgard® ring to filters composed of mixed cellulose esters (Millipore Corp., Bedford, MA). Seeded supports were floated on culture medium and incubated for 11–14 days before being mounted in modified Snapwell holders in Ussing chambers (EasyMount System, Physiologics Instruments, San Diego, CA). Transcellular Cl− flux was then recorded as the short-circuit current as previously described [1Carew M.A. Yang X. Schultz C. Shears S.B. Ins(3,4,5,6)P4 inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell-line.J. Biol. Chem. 2000; 275: 26906-26913Abstract Full Text Full Text PDF PubMed Google Scholar]. The resistance of the monolayers were (vector) 346 ± 44 ohms/cm2 (mean ± SE, n = 12) and (kinase) 354 ± 62 ohms/cm2 (mean ± SE, n = 12). Whole-cell Cl− currents in CFPAC-1 cells were measured at +40mV as previously described, using an intracellular Ca2+-BAPTA buffer to clamp free [Ca2+] to 0.5 μM [8Ho M.W.Y. Kaetzel M.A. Armstrong D.L. Shears S.B. Regulation of a human chloride channel: a paradigm for integrating input from calcium, CaMKII and Ins(3,4,5,6)P4.J. Biol. Chem. 2001; 276: 18673-18680Crossref PubMed Scopus (55) Google Scholar]. American Type Cell Culture (Manassas, VA) supplied the CFPAC-1 cells and T84 cells. Recombinant human Ins(3,4,5,6)P4 1-kinase was expressed in E. coli and purified [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar]. Nonradiolabeled Ins(1,3,4)P3 and Ins(1,3,4,5,6)P5 were purchased from CellSignals Inc (Lexington, KY). The details of the synthesis of nonradiolabeled Ins(3,4,5)P3, Ins(3,5,6)P3, Ins(4,5,6)P3, and Ins(3,4,6)P3 (as sodium salts) will be published separately by S.-K.C. Ins(3,4,6)P3 was also prepared by a different synthetic route [23Mills S.J. Potter B.V.L. Synthesis of D- and L-myo-inositol 1,4,6-trisphosphate, regioisomers of a ubiquitous second messenger.J. Org. Chem. 1996; 61: 8980-8987Crossref PubMed Scopus (27) Google Scholar]. [3H]Ins(1,3,4,5,6)P5 was prepared as previously described [24Zhang T. Caffrey J.J. Shears S.B. The transcriptional regulator, Arg82, is a hybrid kinase with both monophosphoinositol- and diphosphoinositol-polyphosphate synthase activity.FEBS Lett. 2001; 494: 208-212Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. [3H]Ins(1,3,4)P3 was prepared from [3H]Ins(1,3,4,5)P4 (New England Nuclear), using recombinant Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase [25Erneux C. De Smedt F. Moreau C. Rider M. Communi D. Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli. Lack of phosphorylation by protein kinase C.Eur. J. Biochem. 1995; 234: 598-602Crossref PubMed Scopus (14) Google Scholar]. [3H]Ins(1,3,4)P3 was converted to [3H]Ins(1,3,4,6)P4, using Ins(1,3,4)P3 6-kinase activity [4Yang X. Shears S.B. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.Biochem. J. 2000; 351: 551-555Crossref PubMed Scopus (56) Google Scholar]. [3H]Ins(3,4)P2 was prepared by alkaline phosphatase attack on [3H]Ins(1,3,4)P3 in 20 mM glycine (pH 9.0 with KOH). [3H]Ins(1,4)P2 was purchased from New England Nuclear. We thank the Wellcome Trust for Programme Grant support (060554 to B.V.L.P.).
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