The dephosphorylation of inositol 1,4-bisphosphate to inositol in liver and brain involves two distinct Li+-sensitive enzymes and proceeds via inositol 4-phosphate.
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ABSTRACT:
SUBMITTER: Ragan CI
PROVIDER: S-EPMC1148677 | biostudies-other | 1988 Jan
REPOSITORIES: biostudies-other
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Project description:Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.
| S-EPMC1135135 | biostudies-other
Project description:Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.
| S-EPMC1147682 | biostudies-other
Project description:Syntheses of a metabolite of the second messenger myo-inositol 1,4,5-trisphosphate, myo-inositol 1,4-bisphosphate, and an analogue, the 1,4-bisphosphorothioate, are reported, by using phosphite chemistry on (+/-)-1,2:4,5-di-isopropylidene-myo-inositol. The synthesis of (+/-)-1,2:4,5-di-isopropylidene 3,6-bis[di-(2-cyanoethyl)]phosphite provides an intermediate that can be oxidized to either the corresponding bisphosphate or bisphosphorothioate. myo-Inositol phosphorothioates are proposed as novel analogues of myo-inositol phosphates; their resistance to phosphatase-catalysed breakdown is reported.
| S-EPMC1148343 | biostudies-other
Project description:The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.
| S-EPMC1138531 | biostudies-other
Project description:Myofibroblasts are the major cellular source of collagen, and their accumulation - via differentiation from fibroblasts and resistance to apoptosis - is a hallmark of tissue fibrosis. Clearance of myofibroblasts by dedifferentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to dedifferentiate myofibroblasts, but - to our knowledge - the resultant cellular phenotypes have neither been comprehensively characterized or compared. Here, we show that PGE2 elicited dedifferentiation of human lung myofibroblasts via cAMP/PKA, while FGF2 utilized MEK/ERK. The 2 mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast dedifferentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with dedifferentiation occurring in vivo most closely resembling the former.
| S-EPMC8026183 | biostudies-literature
Project description:Muscarinic-receptor-mediated phosphoinositide hydrolysis in rat cerebral cortex was investigated by analysis of the kinetics of [3H]inositol phosphate formation and degradation in myo-[2-3H]inositol-labelled tissue slices. Carbachol stimulated rapid (5 s) increases in the concentrations of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,4)P2. Stimulated accumulation of [3H]Ins(1,3,4)P3, [3H]Ins(1,3)P2 and [3H]Ins(3,4)P2 and [3H]Ins(1/3)P or of [3H]Ins(4)P occurred only subsequently and with a sequence indicating formation by successive dephosphorylation of [3H]Ins(1,3,4,5)P4 or of Ins(1,4)P2 respectively. A similar sequence was inferred from the order of rapidity with which the accumulations of [3H]inositol polyphosphates, resulting from sustained (5 min) carbachol stimulation in the presence of LiCl, were reversed when muscarinic receptors were subsequently blocked with atropine. During this latter period of receptor blockade, radiolabel lost from [3H]inositol polyphosphates was quantitively recovered as [3H]inositol monophosphates owing to effective inhibition of monophosphatase by Li+, and the rate of poly- into mono-phosphate conversion was similar to agonist-stimulated rates of monophosphate accumulation. This implies that, even during persistent stimulation, polyphosphoinositide, not PtdIns, is the substrate for phosphoinositidase C. Quantitative comparison of the degradation of [3H]inositol poly- to mono-phosphates after receptor blockade unexpectedly suggests the dual hydrolysis of [3H]Ins(1,4)P2 to [3H]Ins(1)P and [3H]Ins(4)P. This result advises cautious interpretation of the origin of [3H]Ins(1)P in stimulated tissue, but, with other data presented, allows calculation from the observed ratio of [3H]Ins(1/3)P:[3H]Ins(4)P that a minimum of approx. 50% of the [3H]Ins(1,4,5)P3 produced during persistent muscarinic-receptor stimulation is metabolized by Ins(1,4,5)P3 3-kinase.
| S-EPMC1131959 | biostudies-other
Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].
2013-12-16 | E-MTAB-1528 | biostudies-arrayexpress
Project description:Myosin-I is the single-headed member of the myosin superfamily that associates with acidic phospholipids through its basic tail domain. Membrane association is essential for proper myosin-I localization and function. However, little is known about the physiological relevance of the direct association of myosin-I with phospholipids or about phospholipid headgroup-binding specificity. To better understand the mechanism of myosin-I-membrane association, we measured effective dissociation constants for the binding of a recombinant myo1c tail construct (which includes three IQ domains and bound calmodulins) to large unilamellar vesicles (LUVs) composed of phosphatidylcholine and various concentrations of phosphatidylserine (PS) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). We found that the myo1c-tail binds tightly to LUVs containing >60% PS but very weakly to LUVs containing physiological PS concentrations (<40%). The myo1c tail and not the IQ motifs bind tightly to LUVs containing 2% PIP(2). Additionally, we found that the myo1c tail binds to soluble inositol-1,4,5-trisphosphate with nearly the same affinity as to PIP(2) in LUVs, suggesting that myo1c binds specifically to the headgroup of PIP(2). We also show that a GFP-myosin-I-tail chimera expressed in epithelial cells is transiently localized to regions known to be enriched in PIP(2). Our results suggest that myo1c does not bind to physiological concentrations of PS but rather binds tightly to PIP(2).
| S-EPMC1413866 | biostudies-literature
Project description:Stimulation of [3H]inositol-prelabelled rat cerebral-cortex slices with carbachol results in the accumulation of four [3H]inositol bisphosphate isomeric species, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2 and Ins(4,5)P2. Although the last isomer ran as a minor peak on h.p.l.c., its accumulation was dramatically enhanced in the presence of Li+ (1 mM), such that at 30 min it represented almost 35% of the total bisphosphate fraction. The accumulation of Ins(4,5)P2 appeared to be very sensitive to Li+ (EC50 = 94 +/- 3 microM), strongly implicating a Li(+)-sensitive metabolism. Evidence for this is provided from the rapid but Li(+)-sensitive decay of Ins(4,5)P2 when muscarinic-receptor stimulation is antagonized by atropine at a time when accumulations have reached a new steady state. Manipulation of phospholipase D by activators and inhibitors of protein kinase C did not suggest a role for phospholipase D hydrolysis of PtdInsP2 in the formation of Ins(4,5)P2. Attempts to reveal Ins(4,5)P2 metabolism, or indeed its synthesis from Ins(1,4,5)P3, were not successful with broken cell preparations and strongly suggest discrete compartmentation of inositol phosphate metabolism in the intact cell.
| S-EPMC1133184 | biostudies-other
Project description:The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.
| S-EPMC3293397 | biostudies-literature