Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1.In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells.     

Complete hydrolysis of <Emphasis Type="Italic">myo</Emphasis>-inositol hexakisphosphate by a novel phytase from <Emphasis Type="Italic">Debaryomyces castellii</Emphasis> CBS 2923

Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The K _m value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P₆) to inositol and inorganic phosphate. The sequence of Ins P₆ hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P₅, Ins (1,2,5,6) P₄, Ins (1,2,6) P₃, Ins (1,2) P₂, Ins (1 or 2) P₁, and inositol (notation 3/4/5/6/1 or 2).     

Different Patterns of Agonist-Stimulated Increases of 3H-Inositol Phosphate Isomers and Cytosolic Ca2+ in Bovine Adrenal Chromaffin Cells: Comparison of the Effects of Histamine and Angiotensin II

Abstract: Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP₁, InsP₂, and InsP₃, respectively) isomers, intracellular free Ca²⁺ ([Ca²⁺]_i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca²⁺]_i by 200 nM 3–4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P₃ that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P₃ formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P₃ by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P₂ and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P₃, Ins(1,3)P₂, and Ins(3,4)P₂, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P₂ was the only other InsP₂ besides Ins(1,4)P₂ to accumulate within 1 min of agonist treatment [Ins(3,4)P₂ did not increase]. These results support a correlation between the time course of Ins(1,4,5)P₃ formation and the time course of [Ca²⁺]_i transients and illustrate that Ca²⁺-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca²⁺], changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P₂) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P₃) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P₃-dependent signaling, also negatively regulates PI(4,5)P₂ levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P₃ shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P₂ and PI(3,4,5)P₃, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.     

Metabolism of Inositol(1,4,5)trisphosphate by a Soluble Enzyme Fraction from Pea (Pisum sativum) Roots

Metabolism of the putative messenger molecule d-myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P₃] in plant cells has been studied using a soluble fraction from pea (Pisum sativum) roots as enzyme source and [5-³²P]Ins(1,4,5)P₃ and [2-³H]Ins(1,4,5)P₃ as tracers. Ins(1,4,5)P₃ was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P₂] whereas inositol(1,4)bisphosphate [Ins(1,4)P₂] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P₄. Dephosphorylation of Ins(1,4,5)P₃ to Ins(4,5)P₂ was dependent on Ins(1,4,5)P₃ concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P₃ to Ins(4,5)P₂ and Ins(1,4,5,X)P₄ was inhibited by 55 micromolar Ca²⁺. This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P₃ and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.     

The effect of ischaemia-reperfusion on [3H]inositol phosphates and Ins(1,4,5)P3 levels in cardiac atria and ventricles — a comparative study

In this study incorporation of [³H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P₃ levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [³H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2–3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[³H]Ins(1,4,5)P₃ assay system, the Ins(1,4,5)P₃ levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles.Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P₃ levels as well as incorporation of [3H]inositol into ventricular InSP₂ and InSP₃. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P₃ levels and also [³H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P₃ levels as well as the incorporation of [³H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.     

Histamine-Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis

Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [³H]Inositol 1,4,5-trisphosphate [[³H]Ins(1,4,5)P₃] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of ～140% of basal values. [³H]Inositol 1,4-bisphosphate [[³H]Ins(1,4)P₂] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [³H]inositol 1,3,4-trisphosphate [[³H]-Ins(1,3,4)P₃], [³H]inositol 1,3-bisphosphate, and [³H]-inositol 4-monophosphate ([³H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca²⁺, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [³H]Ins(1,4,5)P₃ was rapidly metabolized (t_1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t_1/2 values of 113, 133, 104, and 66 s for [³H]Ins(1,3,4)P₃, [³H]Ins(1,4)P₂, an unresolved mixture of [³H]inositol 1- and 3-monophosphate ([³H]Ins1/3P), and [³H]Ins4P, respectively. The calculated turnover rate of [³H]Ins(1,4,5)P₃ was sufficient to account for the turnover of the combination of both [³H]Ins(1,4)P₂ and [³H]Ins(1,3,4)P₃ but not that of [³H]Ins1/3P or [³H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca²⁺-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate.     

Inositol phosphate signaling and gibberellic acid

To respond to physical signals and endogenous hormones, plants use specific signal transduction pathways. We and others have previously shown that second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] is used in abscisic acid (ABA) signaling, and that some mutants with altered Ins(1,4,5)P₃ have altered responses to ABA. Specifically, mutants defective in the myo-inositol polyphosphate 5-phosphatases (5PTases) 1 and 2 genes that hydrolyze 5-phosphates from Ins(1,4,5)P₃ and other PtdInsP and InsP substrates, have elevated Ins (1,4,5)P₃, and are ABA-hypersensitive. Given the antagonistic relationship between ABA and gibberellic acid (GA), we tested the response of these same mutants to a GA synthesis inhibitor, paclobutrazol (PAC). We report here that 5ptase1, 5ptase2 and 5ptase11 mutants are hypersensitive to PAC, suggesting a relationship between elevated Ins(1,4,5)P₃ and decreased GA signal transduction. These data provide insight into signaling cross-talk between ABA and GA pathways.Key words: inositol, phosphatidylinositol phosphate, paclobutrazol, gibberellic acid, inositol trisphosphate, paclobutrazol     

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence ⁹¹⁶NFNHIHKRIRRVADKYLSG⁹³⁴ comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence ¹⁴¹⁴SKKTNRGSQLHKYYMKRRTL¹⁴³³, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166–1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.     

Phosphorylated SHIP2 on Y1135 localizes at focal adhesions and at the mitotic spindle in cancer cell lines

The SH2 containing inositol 5-phosphatase SHIP2 is a member of the mammalian phosphoinositide polyphosphate 5-phosphatase family. It is a multi-domain protein comprising a central catalytic domain, an SH2 domain at its N-terminus, proline rich sequences and SAM domain at its C-terminus. It can dephosphorylate both phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and can participate in multiple signaling events in response to growth factors such as EGF, FGF or PDGF. Human SHIP2 can be phosphorylated at two major tyrosine residues Tyr986 and Tyr1135. Here, we report two intracellular localizations of pSHIP2(Y1135): pSHIP2(Y1135)-ir localizes at focal adhesions in EGF-stimulated HeLa cells and at the mitotic spindle in HeLa, in GIST882 cells, a human model of gastrointestinal stromal tumors derived cells, and in human astrocytoma 1321N1 cells. pSHIP2(Y1135)-ir is maximal at metaphase. In N1 cells, evidence is provided that the SHIP2 pathway impacts the distribution of mitotic centrosomes, particularly ү-tubulin. Our data reinforce the concept that SHIP2 localization in intact cells is dependent on phosphorylation mechanisms on both Ser/Thr and Tyr residues, i.e. Y1135, in three cancer cell lines.     

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K⁺ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K⁺ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532–1539). In contrast, transfer to darkness apparently closes open K⁺ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960–962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)P₃ levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P₃ production and K⁺-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P₃ production and K⁺ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P₃ levels and closes K⁺ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K⁺ channels in S. saman pulvinar cells.Abbreviations DiS-C₃-(5) 3,3-dipropylthiadicarbocyanine iodide - F measure change in Dis-C₃-(5) fluorescence - F_o initial Dis-C₃-(5) fluorescence - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate - rbc red blood cell Supported by grants from NSF (IBN 9206179 and MCB 9305154) and U.S.-Israel Binational Agricultural Research and Development Fund (IS-1670-90RC) to R.C.C. We thank the University of Connecticut Biotechnology Center for the use of a fluorescent spectrophotometer.     

Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.     

The behaviour of inositol 1,3,4,5,6-pentakisphosphate in the presence of the major biological metal cations

The inositol phosphates are ubiquitous metabolites in eukaryotes, of which the most abundant are inositol hexakisphosphate (InsP ₆) and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P ₅)]. These two compounds, poorly understood functionally, have complicated complexation and solid formation behaviours with multivalent cations. For InsP ₆, we have previously described this chemistry and its biological implications (Veiga et al. in J Inorg Biochem 100:1800, 2006; Torres et al. in J Inorg Biochem 99:828, 2005). We now cover similar ground for Ins(1,3,4,5,6)P ₅, describing its interactions in solution with Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺ and Fe³⁺, and its solid-formation equilibria with Ca²⁺ and Mg²⁺. Ins(1,3,4,5,6)P ₅ forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe³⁺ is similar to that of InsP ₆ and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P ₅ lacks, is not absolutely necessary for high-affinity Fe³⁺ complexation by inositol phosphates, even if it is necessary for their prevention of the Fenton reaction. With excess Ca²⁺ and Mg²⁺, Ins(1,3,4,5,6)P ₅ also forms the polymetallic complexes [M₄(H₂L)] [where L is fully deprotonated Ins(1,3,4,5,6)P ₅]. However, unlike InsP ₆, Ins(1,3,4,5,6)P ₅ is predicted not to be fully associated with Mg²⁺ under simulated cytosolic/nuclear conditions. The neutral Mg²⁺ and Ca²⁺ complexes have significant windows of solubility, but they precipitate as [Mg₄(H₂L)]·23H₂O or [Ca₄(H₂L)]·16H₂O whenever they exceed 135 and 56 μM in concentration, respectively. Nonetheless, the low stability of the [M₄(H₂L)] complexes means that the 1:1 species contribute to the overall solubility of Ins(1,3,4,5,6)P ₅ even under significant Mg²⁺ or Ca²⁺ excesses. We summarize the solubility behaviour of Ins(1,3,4,5,6)P ₅ in straightforward plots.     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K⁺] above 12 mM and peaked at about 40 mM KCl. In normal calcium buffers, phosphate labeling of PI but not PA declined sharply with [KCl] above 40 mM. In low calcium buffers, the phosphate labeling response was greatly attenuated for both lipids, but PI labeling did not decline at higher [K⁺].The phosphate labeling response was confined to PA and PI, and was specific for the increase in [K⁺]₀. The same response was seen in constant (105 mM) sodium buffers, and atropine had no effect. The specific radioactivity of ATP was increased by elevated potassium, but not enough to account for the increased labeling of PA. Further, this appeared to be a result of the loss of stored ATP rather than an increase in turnover.Increasing [K⁺]₀ produced a decline in [³H]inositol incorporation into PI in parallel with the increase in its labeling by ³³PO₄. This was the same in constant sodium and in low calcium buffers. It could be attributed to an inhibition of synaptosomal uptake of labelled inositol from the medium. Synaptosomal inositol content was unaffected.Elevated potassium had a greater effect on PA labeling than on PI, and it was more effective in increasing phosphate labeling of PA than was acetylcholine (ACh). When ACh and elevated potassium were combined at their maximally effective concentration, they acted synergistically to stimulate phosphate incorporation into PA but elevated potassium blocked the increase in [³H]inositol incorporation into PI normally produced by ACh. These results indicate that elevated potassium and ACh act upon the same population of synaptosomes, but affect different biochemical steps. Elevated potassium probably effects phospholipid labeling by a calcium dependent increase in diglyceride production from lipids other than PA or PI.     

Expression,Purification, Crystallisation and X-ray Crystallographic Analysis of a Truncated Form of Human Src Homology 2 Containing Inositol 5-Phosphatase 2

The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P₃) to form PI(3,4)P₂. PI(3,4,5)P₃ is a key lipid second messenger, which can recruit signalling proteins to the plasma membrane and subsequently initiate numerous downstream signalling pathways responsible for the regulation of a plethora of cellular events such as proliferation, growth, apoptosis and cytoskeletal rearrangements. SHIP2 has been heavily implicated with several serious diseases such as cancer and type 2 diabetes but its regulation remains poorly understood. In order to gain insight into the mechanisms of SHIP2 regulation, a fragment of human SHIP2 containing the phosphatase domain and a region proposed to resemble a C2 domain was crystallized. Currently, no structural information is available on the putative C2-related domain or its relative position with respect to the phosphatase domain. Initial crystals were polycrystalline, but were optimized to obtain diffraction data to a resolution of 2.1 Å. Diffraction data analysis revealed a P2₁2₁2₁ space group with unit cell parameters a = 136.04 Å, b = 175.84 Å, c = 176.89 Å. The Matthews coefficient is 2.54 Å³ Da^?1 corresponding to 8 molecules in the asymmetric unit with a solvent content of 51.7 %.     

“Chelatable iron pool”: inositol 1,2,3-trisphosphate fulfils the conditions required to be a safe cellular iron ligand

Mammalian cells contain a pool of iron that is not strongly bound to proteins, which can be detected with fluorescent chelating probes. The cellular ligands of this biologically important “chelatable”, “labile” or “transit” iron are not known. Proposed ligands are problematic, because they are saturated by magnesium under cellular conditions and/or because they are not “safe”, i.e. they allow iron to catalyse hydroxyl radical formation. Among small cellular molecules, certain inositol phosphates (InsPs) excel at complexing Fe³⁺ in such a “safe” manner in vitro. However, we previously calculated that the most abundant InsP, inositol hexakisphosphate, cannot interact with Fe³⁺ in the presence of cellular concentrations of Mg²⁺. In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3)P ₃], a cellular constituent of unknown function and the simplest InsP to display high-affinity, “safe”, iron complexation. We report thermodynamic constants for the interaction of Ins(1,2,3)P ₃ with Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺ and Fe³⁺. Our calculations indicate that Ins(1,2,3)P ₃ can be expected to complex all available Fe³⁺ in a quantitative, 1:1 reaction, both in cytosol/nucleus and in acidic compartments, in which an important labile iron subpool is thought to exist. In addition, we calculate that the fluorescent iron probe calcein would strip Fe³⁺ from Ins(1,2,3)P ₃ under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3)P ₃. Therefore Ins(1,2,3)P ₃ is the first viable proposal for a transit iron ligand. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: Differential effects ofmyo-inositol

To investigate the effects of increasing concentrations ofmyo-inositol (inositol) on receptor stimulated [³H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [³H]inositol (1 to 30 M). Carbachol stimulated formation of [³H]inositol trisphosphate (InsP₃) and [³H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P₄} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 M inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 M), [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 M inositol, carbachol stimulated [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 M inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [³H]Ins(1,3,4,5)P₄ formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P₄ mass formation by seven fold. Furthermore, Li⁺ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P₄ mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P₄. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li⁺, increasing the inositol concentration greatly increases the stimulated component of [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P₄ formation is not reflected as an increase in mass formation.     

Carbachol- and KCl-induced changes in phosphoinositide metabolism and free calcium in guinea pig cerebral cortex synaptosomes

Phosphoinositide (PI) and calcium metabolism were studied in guinea pig cerebral cortex synaptosomes. Mass amounts of inositol and inositol monophosphates, and the levels of free intrasynaptosomal calcium ([Ca²⁺]_i) were measured after KCl (60 mM), after a direct cholinergic agonist carbachol (CA, 1mM), and after their combination. Inositol, inositol-1-phosphate (Ins1P), inositol-4-phosphate (Ins4P) and [Ca²⁺]_i were measured with and without 10 mM LiCl in the incubation medium. CA-induced cholinergic stimulation elevated synaptosomal Ins4P levels by 40% but did not affect Ins1P or [Ca²⁺]_i. On the contrary, KCl elevated Ins1P by 50% and [Ca²⁺]_i by 40% above the resting level, and decreased inositol by 20%, whereas no alterations in Ins4P occurred. CA did not modify the response of KCl, but KCl abolished the elevation of Ins4P by CA. LiCl attenuated KCl-induced elevation of Ins1P but amplified the CA-induced elevation of Ins4P. The elevation of presynaptic [Ca²⁺]_i was accompanied by accumulation of Ins1P but not that of Ins4P. Hence, the present results suggest that presynaptic cholinergic stimulation and KCl-induced depolarization may activate different degradation pathways of inositolphosphate metabolism.