Hydrolysis of phosphatidylinositol 3,4-bisphosphate by inositol polyphosphate 4-phosphatase isolated by affinity elution chromatography

Inositol polyphosphate 4-phosphatase is a monomeric 110-kDa protein that hydrolyzes two substrates in the inositol phosphate pathway. Inositol 3,4-bisphosphate is converted to inositol 3-phosphate, and inositol 1,3,4-trisphosphate is converted to inositol 1,3-bisphosphate. We have exploited the fact that inositol hexasulfate inhibits the enzyme to devise an affinity elution scheme from a Mono S cation exchange column that resulted in an 11,300-fold purified preparation of rat brain 4-phosphatase. The resulting 4-phosphatase hydrolyzed phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3-phosphate with a first order rate constant 120-fold greater than that for inositol 3,4-bisphosphate and 900-fold greater than that for inositol 1,3,4-trisphosphate. This is now the third example wherein the same enzyme hydrolyzes both an inositol lipid and its analogous inositol phosphate.     

The cDNA cloning and characterization of inositol polyphosphate 4-phosphatase type II. Evidence for conserved alternative splicing in the 4-phosphatase family

Inositol polyphosphate 4-phosphatase (4-phosphatase) is a Mg2+-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 3,4-bisphosphate. We have isolated cDNA encoding a 105,257-Da protein that is 37% identical to the previously cloned 4-phosphatase. Recombinant protein was expressed in Escherichia coli and shown to hydrolyze all three 4-phosphatase substrates with enzymatic properties similar to the original enzyme. We designate the original 4-phosphatase and the new isozyme as inositol polyphosphate 4-phosphatase types I and II, respectively. 4-Phosphatase II is highly conserved with the human and rat enzymes having 90% amino acid identity. A conserved motif between 4-phosphatase I and II is the sequence CKSAKDRT that contains the Cys-Xaa5-Arg active site consensus sequence identified for other Mg2+-independent phosphatases. Northern blot analysis indicated that 4-phosphatase II is widely expressed with the highest levels occurring in the skeletal muscle and heart. In addition, cDNA encoding alternatively spliced forms of human 4-phosphatase I (107, 309 Da) and rat 4-phosphatase II (106,497 Da) were also isolated that encode proteins with a putative transmembrane domain near their C termini. These alternatively spliced forms were expressed as recombinant proteins in E. coli and SF9 insect cells and found to possess no detectable enzymatic activity suggesting that additional factors and/or processing may be required for these alternatively spliced isozymes.     

The SH2-containing inositol polyphosphate 5-phosphatase, ship, is expressed during hematopoiesis and spermatogenesis

Ship is a recently identified SH2-containing inositol polyphosphate 5-phosphatase that has been implicated as an important signaling molecule in cell-culture systems. To understand the physiologic function of Ship in vivo, we performed expression studies of Ship during mouse development. Results of this study demonstrate the expression of ship to be in late primitive-streak stage embryos (7.5 days postcoitus [dpc]), when hematopoiesis is thought to begin, and the expression is restricted to the hematopoietic lineage in mouse embryo. In adult mice, Ship expression continues to be in the majority of cells from hematopoietic origin, including granulocytes, monocytes, and lymphocytes, and is also found in the spermatids of the testis. Furthermore, the level of Ship expression is developmentally regulated during T-cell maturation. These results suggest a possible role for Ship in the differentiation and maintenance of the hematopoietic lineages and in spermatogenesis.     

The inositol polyphosphate 5-phosphatase ship is a crucial negative regulator of B cell antigen receptor signaling

Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcgamma receptor IIB (FcgammaRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)-FcgammaRIIB coligation. The function of Ship in lymphocytes was investigated in Ship-/- recombination-activating gene (Rag)-/- chimeric mice generated from gene-targeted Ship-/- embryonic stem cells. Ship-/-Rag-/- chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship-/- splenic B cells displayed prolonged Ca2+ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR-FcgammaRIIB coligation. These results demonstrate that Ship plays an essential role in FcgammaRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca2+ flux and MAPK activation.     

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

INP51, a yeast inositol polyphosphate 5-phosphatase required for phosphatidylinositol 4,5-bisphosphate homeostasis and whose absence confers a cold-resistant phenotype

Sequence analysis of Saccharomyces cerevisiae chromosome IX identified a 946 amino acid open reading frame (YIL002C), designated here as INP51, that has carboxyl- and amino-terminal regions similar to mammalian inositol polyphosphate 5-phosphatases and to yeast SAC1. This two-domain primary structure resembles the mammalian 5-phosphatase, synaptojanin. We report that Inp51p is associated with a particulate fraction and that recombinant Inp51p exhibits intrinsic phosphatidylinositol 4,5-bisphosphate 5-phosphatase activity. Deletion of INP51 (inp51) results in a "cold-tolerant" phenotype, enabling significantly faster growth at temperatures below 15 degreesC as compared with a parental strain. Complementation analysis of an inp51 mutant strain demonstrates that the cold tolerance is strictly due to loss of 5-phosphatase catalytic activity. Furthermore, deletion of PLC1 in an inp51 mutant does not abrogate cold tolerance, indicating that Plc1p-mediated production of soluble inositol phosphates is not required. Cells lacking INP51 have a 2-4-fold increase in levels of phosphatidylinositol 4,5-bisphosphate and inositol 1,4, 5-trisphosphate, whereas cells overexpressing Inp51p exhibit a 35% decrease in levels of phosphatidylinositol 4,5-bisphosphate. We conclude that INP51 function is critical for proper phosphatidylinositol 4,5-bisphosphate homeostasis. In addition, we define a novel role for a 5-phosphatase loss of function mutant that improves the growth of cells at colder temperatures without alteration of growth at normal temperatures, which may have useful commercial applications.     

The diversity and possible functions of the inositol polyphosphate 5-phosphatases

Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates, i.e., inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), inositol 1,3,4, 5-tetrakisphosphate (Ins(1,3,4,5)P4), phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) or phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P3). In mammalian cells, this family contains a series of distinct genes and splice variants. All inositol polyphosphate 5-phosphatases share a 5-phosphatase domain and various protein modules probably responsible for specific cell localisation or recruitment (SH2 domain, proline-rich sequences, prenylation sites, etc.). Type I Ins(1,4,5)P3 5-phosphatase also uses Ins(1,3,4,5)P4 but not the phosphoinositides as substrates. This enzyme is targeted to specific membranes by means of a prenylation site. Type II 5-phosphatases can use both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as substrates. Five mammalian enzymes and multiple splice variants are known: INPP5P or inositol polyphosphate 5-phosphatase II, OCRL (a Golgi protein implicated in the Lowe oculocerebrorenal syndrome), synaptojanin (a protein involved in the recycling of synaptic vesicles), SHIP 1 and SHIP 2 (or SH2-containing inositol 5-phosphatases). As discussed in this review, the substrate specificity, regulatory mechanisms, subcellular localisation and tissue specificity indicate that the different 5-phosphatase isoforms may play specific roles. As known in the dephosphorylation of tyrosine containing substrates by the tyrosine protein phosphatases or in the metabolism of cyclic nucleotides by the cyclic nucleotide phosphodiesterases, inositol polyphosphate 5-phosphatases directly participate in the control of second messengers in response to both activation or inhibitory cell signalling.     

Attenuation of LPA-mediated calcium signaling and inositol polyphosphate production in rat-1 fibroblasts transformed by the v-src oncogene

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]i and PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i (EC50 = 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrc cells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3 and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2 in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2 in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.     

Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants

To investigate molecular and clinical aspects of conotruncal anomaly face (CAF), we studied the correlation between deletion size and phenotype and the mode of inheritance in 183 conotruncal anomaly face syndrome (CAFS) patients. Hemizygosity for a region of 22ql1.2 was found in 180 (98%) of the patients with CAFS by fluorescence in situ hybridization (FISH) using the N25(D22S75) DiGeorge critical region (DGCR) probe. No hemizygosity was found in three (2%) of the patients with CAFS by FISH using nine DiGeorge critical region probes and a SD1OP1 probe (DGA II locus). None of these three patients had mental retardation and just one had nasal intonation, which was observed in almost all of the 180 CAFS patients who carried deletions (mental retardation, 92%; nasal voice, 88%). Nineteen of 143 families (13%) had familial CAFS and 16 affected parents (84%) were mothers. Although only two of the affected parents had cardiovascular anomalies, the deletion size in the 16 affected parents and their affected family members, who were studied by FISH analysis, was the same. It indicates that extragenic factors may play a role in the genesis of phenotypic variability, especially in patients with cardiovascular anomalies. No familial cases were found among CAFS patients with absent thymus/DiGeorge anomaly (DGA). Also, in all 18 CAFS patients with completely absent thymus/DGA and all 6 CAFS patients with schizophrenia, it was revealed that the deletion was longer distally. A study of the origin of the deletion using microsatellite analyses in 48 de novo patients showed that in 65% of CAFS patients it was maternal, while in 64% of DGA patients it was paternal. The findings of this study indicated that CAF was almost always associated with the deletion of 22ql1.2. As well as the major features of the syndrome, other notable extracardiac anomalies were found to be susceptibility to infection, schizophrenia, atrophy or dysmorphism of the brain, thrombocytopenia, short stature, facial palsy, anal atresia, and mild limb abnormalities.     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutation chloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutant fer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that the fer gene is epistatic over the chln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. The chln gene is located on chromosome 1 and the fer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate the fer gene by map-based cloning. The isolation of the fer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

The in vivo regulation of liver and kidney glucose-6-phosphatase by dexamethasone

The microsomal glucose-6-phosphatase enzyme is situated with its active site inside the lumen of the endoplasmic reticulum and for normal enzyme activity in vivo, transport systems are needed for the substrates and products of the enzyme. Most studies of glucose-6-phosphatase have been carried out on the liver enzyme and relatively little is known about the regulation of the kidney glucose-6-phosphatase enzyme system. Here we demonstrate that the liver and kidney glucose-6-phosphatase systems are regulated differently by dexamethasone and that dexamethasone acts on both the glucose-6-phosphatase enzyme and T1 its associated glucose-6-phosphate transport protein.     

Chronic ethanol inhibits inositol metabolism in specific brain regions

Many neurotransmitters and hormones in the nervous system transmit signals through receptors coupled to the poly-phosphoinositide (PI) signaling pathway. In this study, an in vivo protocol with [3H]inositol was used to examine the effect of chronic ethanol administration on inositol metabolism and poly-PI turnover in the cerebral cortex, hippocampus, and cerebellum of mouse brain. C57BL/6 mice were given a nutritionally complete liquid diet containing either ethanol (5%, w/v) or isocaloric sucrose for 2 months. Mice were injected intracerebrally with [3H]inositol; after 16 or 24 hr, they were injected intraperitoneally with lithium (8 mEq/kg body weight) to inhibit the inositol monophosphatase (IP1) activity. All mice were decapitated 4 hr after lithium injection. Labeled inositol phospholipids accounted for 16 to 23% of total labeled inositol in different regions of control mouse brain, and the percentages in the hippocampus were consistently higher than the cerebral cortex and cerebellum. In control mice, the percentages of labeled IP1 after a 4-hr lithium treatment were 11.5%, 9.9%, and 3.7% for cerebral cortex, hippocampus, and cerebellum, respectively. Chronic ethanol feeding resulted in a significant (p < 0.05) decrease in the percent of labeled IP1 and inositol phospholipids, and this effect was observed in the cerebral cortex and, to a lesser extent, hippocampus but not cerebellum. When ratios of labeled IP1 were expressed against labeled inositol phospholipids as an index of the poly-PI turnover activity, significant decreases in IP/lipid ratios were observed in the cerebral cortex, but not the hippocampus or cerebellum. Although mice killed 24 + 4 hr after the last ethanol feeding would have experienced an 8-hr period of ethanol withdrawal, compared with the 16 + 4-hr group, no differences in IP/lipid ratios were observed between the two time groups. These results illustrate regional differences in the effect of chronic ethanol on inositol metabolism in the brain, but no difference in poly-PI turnover in brain due to ethanol withdrawal.     

Catalytic defects in mutants of class II histidyl-tRNA synthetase from Salmonella typhimurium previously linked to decreased control of histidine biosynthesis regulation

The expression of histidine biosynthetic genes in enteric bacteria is regulated by an attenuation mechanism in which the level of histidyl-tRNA serves as a key sensor of the intracellular histidine pool. Among the early observations that led to the formation of this model for Salmonella typhimurium were the identification of mutants in the gene (hisS) encoding histidyl-tRNA synthetase. We report here the detailed biochemical characterization of five of these S. typhimurium bradytrophic mutants isolated by selection for resistance to histidine analogs, including identification of the deduced amino acid substitutions and determination of the resulting effects on the kinetics of adenylation and aminoacylation. Using the crystal structure of the closely related Escherichia coli histidyl-tRNA synthetase (HisRS) as a guide, two mutants were mapped to a highly conserved proline residue in motif 2 (P117S, P117Q), and were correlated with a fivefold decrease in the kcat for the pyrophosphate exchange reaction, as well as a tenfold increase in the Km for tRNA in the aminoacylation reaction. Another mutant substitution (A302T) mapped to a residue adjacent to the histidine binding pocket, leading to a tenfold increase in Km for histidine in the pyrophosphate exchange reaction. The remaining two mutants (S167F, N254T) substitute residues in or directly adjacent to the hinge region, which joins the insertion domain between motif 2 and motif 3 to the catalytic core, and cause the Km for tRNA to increase four- to tenfold. The kinetic analysis of these mutants establishes a direct link between critical interactions within the active site of HisRS and regulation of histidine biosynthesis, and provides further evidence for the importance of local conformational changes during the catalytic cycle.     

Synaptic physiology and ultrastructure in comatose mutants define an in vivo role for NSF in neurotransmitter release

N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Our previous work showed that mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, we perform electrophysiological and ultrastructural analyses in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses revealed that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures revealed a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, we used transmission electron microscopy to examine the distribution of vesicles within synaptic terminals, and observed a marked accumulation of docked vesicles at restrictive temperatures in comt. Together, the results reported here define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion.     

Differential induction of inositol phosphate metabolism by three adipokinetic hormones

Sheep naturally infected by visna-maedi virus often develop a chronic interstitial lung disease characterized by an alveolitis comprising lymphocytes, neutrophils and macrophages. The alpha chemokine interleukin-8 (IL-8) was detected in cell free bronchoalveolar lavage fluid from naturally infected animals, confirmed by RT-PCR, presenting typical lesions of maedi and elevated total alveolar cell counts. No detectable IL-8 was found in the fluid obtained from uninfected animals. IL-8 concentration in alveolar fluid is correlated with alveolar neutrophil counts. Bronchoalveolar lavage cells from infected animals were found to contain a large amount of IL-8 mRNA and may contribute to IL-8 production. In situ hybridization showed that macrophages were the predominant cell type expressing IL-8 mRNA. Sustained production of IL-8 by alveolar macrophages during visna-maedi infection could suffice for neutrophil attraction to the alveoli, and may contribute to the development of lesions.