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PubMed1997

Activation of the 43 kDa inositol polyphosphate 5-phosphatase by 14-3-3zeta.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyzes and thereby inactivates the second messenger molecules inositol 1,4,5-trisphosphate -Ins(1,4,5)P3- and inositol 1,3,4,5-tetrakisphosphate in a signal terminating reaction. Recent studies have shown that the platelet protein pleckstrin forms a complex with the 43 kDa 5-phosphatase and activates Ins(1,4,5)P3 hydrolysis 2-fold [Auethavekiat, V., Abrams, C. S., & Majerus, P. W. (1997) J. Biol. Chem. 272, 1786-1790]. We now show that another platelet protein, 14-3-3zeta, forms a complex with the 43 kDa 5-phosphatase and thereby activates the hydrolysis of Ins(1,4,5)P3. Both pleckstrin and 14-3-3zeta contain one or more pleckstrin-homology domains, both are present in platelet cytosol, and both dimerize and form complexes with other signalling proteins. Purified platelet pleckstrin and 14-3-3zeta enhanced the rate of the hydrolysis of Ins(1,4,5)P3 by the 43 kDa 5-phosphatase 1.9- and 3.8-fold, respectively, but did not activate the 75 kDa 5-phosphatase. We have demonstrated that the mechanism of 5-phosphatase activation by 14-3-3zeta results from specific complex formation between the 43 kDa 5-phosphatase and 14-3-3zeta. Recombinant 43 kDa 5-phosphatase bound to recombinant glutathione S-transferase (GST)/14-3-3zeta fusion protein, but not GST alone, immobilized on glutathione-Sepharose. A potential 14-3-3 binding motif was located in the 43 kDa, but not the 75 kDa, 5-phosphatase. The motif "363RSESEE" is present in close proximity to the proposed catalytic domain of the 43 kDa 5-phosphatase. A synthetic peptide corresponding to the putative 14-3-3 binding motif demonstrated specific, saturable binding to purified 125I-14-3-3, with a Kd of 92 nM. In addition, platelet cytosolic 5-phosphatase bound to recombinant 14-3-3zeta immobilized on glutathione-Sepharose. Thus, 14-3-3zeta serves in human platelets to activate the 43 kDa 5-phosphatase and may thereby function to prevent generation of Ins(1,4,5)P3 -mediated calcium release in unstimulated platelets.

14-3-3 Proteins↗
PubMedDate not supplied

Novel biodegradable polyphosphate cross-linker for making biocompatible hydrogel.

To obtain a novel biodegradable cross-linker, polymerizable polyphosphate (PIOP) was synthesized by ring-opening polymerization of 2-i-propyl-2-oxo-1,3,2-dioxaphospholane with 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl methacrylate) (OPEMA). The number averaged molecular weight of the PIOP was 1.2 x 10(4), and the number of OPEMA units in one PIOP molecule was 2.2. Nonenzymatic degradation of the PIOP was evaluated in various pH aqueous media. The degree of hydrolysis was dependent on the pH; that is, it increased with an increase in the pH of the medium. At pH 11.0, the PIOP completely degraded in only 6 days. The poly[2-methacryloyloxyethyl phosphorylcholine (MPC)] cross-linked with the PIOP was prepared by radical polymerization. This polymer could form hydrogel, and the free water fraction in the hydrogel was high. The enzymatic activity of trypsin in contact with the hydrogel was similar to that in buffer solution. There is no adverse effect caused by the hydrogel to reduce the function of the trypsin. The cytotoxicity of poly(MPC) and degraded PIOP was evaluated using v79 cells, and it was not observed in either case. In conclusion, PIOP is a hydrolyzable polymer, which can be used as a cross-linker, and novel hydrogels having biodegradability and biocompatibility were prepared from poly(MPC) cross-linked with the PIOP.

Animals↗
PubMed2002

The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173.

The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is approximately 15% of the total cellular polyP. Over 80% of the vacuole polyP are in an acid-soluble fraction. It was first established by (31)P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba(2+) in succession at pH 4.5 and 8.2 was approximately 20 +/- 5 and 5 +/- 2 residues of ortho-phosphoric acid, respectively. Under a deficit of phosphate (P(i)) in the cultivation medium, the polyP content in vacuoles decreased approximately 7-fold with the same drastic reduction of their content in the cell. Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect. The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell.

Hydrogen-Ion Concentration↗
PubMed1992

The Lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase.

Lowe's oculocerebrorenal syndrome (OCRL) is a human X-linked developmental disorder of unknown pathogenesis and has a pleiotropic phenotype affecting the lens, brain and kidneys. The OCRL locus has been mapped to Xq25-q26 by linkage and by finding de novo X; autosome translocations at Xq25-q26 in two unrelated females with OCRL. Here we use yeast artificial chromosomes with inserts that span the X chromosomal breakpoint from a female OCRL patient in order to isolate complementary DNAs for a gene that is interrupted by the translocation. We show that the transcript is absent in both female OCRL patients with X; autosome translocations and that it is absent or abnormally sized in 9 of 13 unrelated male OCRL patients with no detectable genomic rearrangement. The open reading frame encodes a new protein with 71% similarity to human inositol polyphosphate-5-phosphatase. Our results suggest that OCRL may be an inborn error of inositol phosphate metabolism.

Amino Acid Sequence↗
PubMed2001

Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P(2), forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P(2) in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P(2) 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P(2) levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P(2) levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for Deltainp51Deltainp53, Deltainp51Deltainp52 and Deltainp52Deltainp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P(2) accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P(2) 5-phosphatases.

Animals↗
PubMed1996

Regulation of second messengers by the inositol polyphosphate 5-phosphatases.

The inositol polyphosphate 5-phosphatases are an enlarging family of enzymes that terminate the signals generated by the phosphoinositide kinases and phospholipase C. Given the diverse signalling functions of both the polyphosphoinositides and Ins(1,4,5)P3, it is predicted that the 5-phosphatases will play a critical role in regulating many cellular events, in particular membrane trafficking and cell growth.

Amino Acid Sequence↗
PubMed1997

Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate.

BACKGROUND: RNA polymerase from the stationary growth phase cells of Escherichia coli is tightly associated with an acidic compound(s) and exhibits altered promoter selectivity, with reduced transcriptional activity of the genes highly expressed in the exponentially growing cells. Here we have examined the nature of the RNA polymerase-associated acidic compound(s). RESULTS: RNA polymerase isolated from stationary-phase cells of E. coli was found to be tightly associated with inorganic polyphosphates (poly P), and cannot be dissociated even after chromatography on phosphocellulose or DNA-cellulose columns. Since RNA polymerase-poly P complexes reconstituted in vitro showed similar properties, poly P was not binding covalently. The inhibitory effects of poly P on transcription were examined using two forms of the holoenzyme, one containing sigma70, the major sigma for transcription of the genes expressed during exponential cell growth and the other containing sigma38, the principal sigma in the stationary growth phase. At low salt concentrations, poly P inhibited transcription by both Esigma70 and Esigma38 holoenzymes. With an increase in the concentration of potassium glutamate, the poly P inhibition was relieved. At high salt concentrations, the Esigma70 holoenzyme is not able to function, but the Esigma38 holoenzyme is however activated. CONCLUSIONS: These results show that poly P may play a role in the promoter selectivity control of RNA polymerase in E. coli which is growing under conditions of high osmolarity and in the stationary growth phase.

Chromatography↗
PubMed1998

Alginate, inorganic polyphosphate, GTP and ppGpp synthesis co-regulated in Pseudomonas aeruginosa: implications for stationary phase survival and synthesis of RNA/DNA precursors.

The regulatory protein AlgR2 in Pseudomonas aeruginosa positively regulates nucleoside diphosphate kinase (Ndk) and succinyl-CoA synthetase, enzymes critical in nucleoside triphosphate (NTP) formation. AlgR2 positively regulates the production of alginate, GTP, ppGpp and inorganic polyphosphate (poly P). An algR2 mutant with low levels of these metabolites has them restored by introducing and overexpressing either the algR2 or the ndk gene into the algR2 mutant. Thus, Ndk is involved in the formation of these compounds and largely prevents the death of the algR2 mutant, which occurs early in the stationary phase. We demonstrate that the 12 kDa Ndk-pyruvate kinase (Pk) complex, previously shown to generate predominantly GTP instead of all the NTPs, has a low affinity for the deoxynucleoside diphosphates and cannot generate the dNTPs needed for DNA replication and cell division; this complex may thus be involved in regulating the levels of both NTPs and dNTPs that modulate cell division and survival in the stationary phase.

Alginates↗
PubMed1998

IMP-GMP 5'-nucleotidase from rat brain: activation by polyphosphates.

IMP-GMP 5'-nucleotidase has been purified to homogeneity from total rat brain extracts. This preparation showed a unique band (Mr 54,000 +/- 1,509) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme presented the following properties: optimal pH value, 6.5-6.8; relative velocity measured in the presence of MgCl2, MnCL2, CoCl2, and NiCl2 (2 mM), 100, 60, 11, and <1, respectively; preferred substrates, IMP and GMP; and activation constant (Ka) found for Ap4A, Ap5A, and Ap6A, 83 +/- 38, 77 +/- 32, and 57 +/- 12 microM, respectively. Under assay conditions where activation by Ap4A was fivefold, the activation produced by dinucleotides was as follows: Ap4G (4.0), Ap4I (2.9), Ap4X (3.3), Ap4C (0.7), Ap4U (1.1), Ap4epsilonA (1.5), Ap4ddA (1.7), Gp4G (2.2), Ap3A (1.1), and Ap2A (1.2). Polyphosphates P18, P19, P20, and P35 were activators of the reaction with calculated Ka values of 3.5 +/- 0.5, 0.9 +/- 0.2, 0.6 +/- 0.2, and 1.3 +/- 0.5 microM, respectively. The following compounds, at 0.1 mM, were effectors of the phosphotransferase reaction producing the fold activation indicated: Ap4A (8.3), Ap5A (10.2), Ap6A (10.1), Ap4G (7.7), Ap4X (7.6), Ap4U (2.1), glycerate 2,3-bisphosphate (3.9), and unpurified P15 (7.6). Two enzyme forms of IMP-GMP 5'-nucleotidase were detected when the extracts from rat tissues or from the crustacean Artemia were subjected to chromatography on a Dyematrex Green A column. The ratio of the hydrolytic activities under both peaks (peak I/peak II) was as follows: brain (1.5), heart (1.9), liver (1.6), lung (2.0), testis (3.8), and Artemia cysts (2.0).

5'-Nucleotidase↗
PubMed2004

Inorganic polyphosphate in the origin and survival of species.

Inorganic polyphosphate (poly P), in chains of tens to hundreds of phosphate residues, linked by high-energy bonds, is environmentally ubiquitous and abundant. In prebiotic evolution it could have provided a flexible, polyanionic scaffold to assemble macromolecules. It has been conserved in every cell in nature. In prokaryotes, a major poly P synthetic enzyme is poly P kinase 1 (PPK1), which is found in 100 bacterial genomes, including numerous pathogens. Null mutants of PPK1, with low poly P levels, are defective in survival: namely, they show defective responses to physical/chemical stresses and predation. Pathogens with a PPK1 deletion are defective in biofilm formation, quorum sensing, general stress and stringent responses, motility, and other virulence properties. With the exception of Dictyostelium, PPK1 is absent in eukaryotes and provides a novel target for chemotherapy that would affect both virulence and susceptibility to antibacterial compounds. Remarkably, another PPK in Dictyostelium discoideum (PPK2) is an actin-related protein (Arp) complex that is polymerized into an actin-like filament, concurrent with its reversible synthesis of a poly P chain from ATP.

Adenosine Triphosphate↗
PubMed2004

Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate.

Inorganic polyphosphate (poly P), a chain of hundreds of phosphate residues linked by ATP-like bonds, is found in every cell in nature and is commonly produced from ATP by poly P kinases (e.g., PPK1). Dictyostelium discoideum, the social slime mold, possesses a PPK activity (DdPPK1) with sequence similarity to bacterial PPKs. We find here a previously unrecognized PPK (DdPPK2) in D. discoideum with the sequences and properties of actin-related proteins (Arps) that are similar to muscle actins in size, properties, and globular-filamentous structural transitions. Significantly, the unique actin inhibitors, phalloidin and DNase I, also inhibit synthesis of poly P by DdPPK2. Thus, this particular Arp complex is an enzyme that can polymerize into an actin-like filament concurrent with its synthesis of a poly P chain in a fully reversible reaction.

Actins↗
PubMed2004

Inorganic polyphosphate in Bacillus cereus: motility, biofilm formation, and sporulation.

Chains of inorganic polyphosphate (poly-P) with hundreds of P(i) residues linked by phosphoanhydride bonds, as in ATP, are found in every bacterial, fungal, plant, and animal cell, in which they perform various functions. In the spore-forming Bacillus cereus, we have identified three principal enzymes and genes involved in the metabolism of poly-P, namely, (i) poly-P kinase (PPK), which synthesizes poly-P reversibly from ATP, (ii) exopolyphosphatase (PPX), which hydrolyzes poly-P to P(i), and (iii) poly-P/AMP phosphotransferase (PAP), which uses poly-P as a donor to convert AMP to ADP, reversibly. In the null mutant of ppk, poly-P levels are reduced to <5% of the WT; in the ppx mutant, the PPK activity is elevated 10-fold, and the accumulation of poly-P is elevated approximately 1,000-fold. All of the null mutants of ppk, ppx, and pap showed defects in motility and biofilm formation, but sporulation efficiency was impaired only in the ppx mutant. These enzymes and genes in B. cereus are nearly identical to those in the very closely related pathogen Bacillus anthracis, and, thus, they may provide attractive targets for the treatment of anthrax.

Acid Anhydride Hydrolases↗
PubMed2005

Inorganic polyphosphate in Dictyostelium discoideum: influence on development, sporulation, and predation.

Dictyostelium discoideum, a social slime mold that forms fruiting bodies with spores, depends on inorganic polyphosphate (poly P) for its cycles of development and for nutritional predation on bacteria. The synthesis of poly P, a polymer of tens or hundreds of phosphate residues linked by high energy, ATP-like bonds, is catalyzed in most bacteria by poly P kinase (PPK1). The eukaryote D. discoideum possesses a homolog of PPK1. We report here that mutants of D. discoideum PPK1 (DdPPK1) have reduced levels of poly P and are deficient in development. Fruiting bodies are smaller and produce fewer spores, which appear to germinate like the wild type (WT). The DdPPK1 mutant formed smaller plaques on bacterial lawns compared with those of the WT. Predation by D. discoideum, assessed by uptake and digestion of Klebsiella aerogenes, showed that fewer bacteria were taken up by the DdPPK1 mutant compared with the WT and were killed less rapidly, indicating a role of poly P and/or DdPPK1 in phagocytosis. On Pseudomonas aeruginosa lawns, cleared plaques were observed with the bacterial PPK1 mutant but not with the WT P. aeruginosa. Thus, poly P is important in predation both for the predator and prey.

Animals↗
PubMed2005

Inorganic polyphosphate in the social life of Myxococcus xanthus: motility, development, and predation.

Inorganic polyphosphate (poly P), a polymer of tens or hundreds of phosphate residues linked by high-energy, ATP-like bonds, is found in all organisms and performs a wide variety of functions. Myxococcus xanthus, a social bacterium that feeds on other bacteria and forms fruiting bodies and spores, depends on poly P for motility, development, and nutritional predation. Two poly P metabolizing enzymes were studied in M. xanthus: poly P kinase 1, which synthesizes poly P reversibly from ATP, and poly P:AMP phosphotransferase, which uses poly P as a donor to also reversibly convert AMP to ADP. The null mutant of ppk1 is defective in social motility, overproduces pilin protein on the cell surface, is delayed in fruiting body formation, produces fewer spores, is delayed in germination, and forms far smaller plaques on a lawn of Klebsiella aerogenes. The pap mutant is also impaired in social motility, but shows only slightly reduced abilities in development and predation.

Adenine Nucleotides↗
PubMed2002

Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

The importance of inorganic polyphosphate (poly P) and poly P kinase (PPK), the enzyme principally responsible for its synthesis, has been established previously for stationary-phase survival of Escherichia coli and virulence in Pseudomonas aeruginosa. The gene (ppk) that encodes PPK is highly conserved among many bacterial pathogens, including Shigella and Salmonella spp. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed in stationary phase, the ppk gene of pathogenic Shigella flexneri, Salmonella enterica serovar Dublin, and Salmonella enterica serovar typhimurium have been cloned and deleted. In some of these mutants lacking ppk, the phenotypes included features indicative of decreased virulence such as: (i) growth defects, (ii) defective responses to stress and starvation, (iii) loss of viability, (iv) polymyxin sensitivity, (v) intolerance to acid and heat, and (vi) diminished invasiveness in epithelial cells. Thus PPK may prove, as it has with P. aeruginosa, to be an attractive target for antibiotics, with low toxicity because PPK is not found in higher eukaryotes.

Aerobiosis↗
PubMed1995

Properties of type II inositol polyphosphate 5-phosphatase.

We have isolated additional cDNA clones encoding type II inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a combined cDNA of 3076 nucleotides encoding a protein of 942 amino acids. The 5-phosphatase II hydrolyzed both Ins(1,4,5)P3 to Ins(1,4)P2 and the phospholipid PtdIns(4,5)P2 to PtdIns(4)P both in vitro and in vivo. There are two motifs highly conserved between types I and II 5-phosphatase and several other proteins presumed to be inositol phosphatases suggesting a possible role in catalysis. The type II 5-phosphatase also contains homology to several GTPase activating proteins although no such activity for 5-phosphatase II was found. The predicted protein ends with the sequence CNPL, suggesting that it is isoprenylated as a mechanism for membrane attachment. We found evidence for isoprenylation by demonstrating incorporation of [3H]mevalonate into native but not C939S mutant 5-phosphatase II expressed in Sf9 insect cells. Furthermore, we showed that membrane localization and the activity of 5-phosphatase II toward its lipid substrate PtdIns(4,5)P2 is reduced by eliminating 5-phosphatase II isoprenylation in the mutant C939S relative to the native enzyme.

Amino Acid Sequence↗
PubMed1995

Inorganic polyphosphates in the acquisition of competence in Escherichia coli.

A complex of polyhydroxybutyrate (PHB), Ca2+, and inorganic polyphosphate (polyP) was proposed as the membrane component responsible for competence for DNA entry in Escherichia coli (Reusch, R. N., and Sadoff, H. L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4176-4180). While chemical and immunological assays and 1H NMR have unequivocally established the identity and content of PHB in the complex, comparable methods were not available for polyP. With specific enzyme assays developed for polyP, we have identified, in chloroform extracts of competent cell membranes, a novel form of polyP of about 60 to 70 residues in a stoichiometric ratio of PHB to polyP of 2:1. In E. coli mutants, incapable of synthesizing the predominant, thousand-long polyP chains, appearance of this short polyP and its inclusion in membranes can account for their capacity to develop competence and indicates an auxiliary pathway for polyP synthesis. A variety of fluorescent lipid probes demonstrate the appearance of extensive rigid domains in membranes of competent cells. We propose that the PHB.Ca2+.polyP complex perturbs the conformation of the lipid matrix, making it more permeable to charged molecules and thus allowing the entry of DNA.

Cell Membrane↗
PubMed1996

Endopolyphosphatases for long chain inorganic polyphosphate in yeast and mammals.

Whereas exopolyphosphatases have been purified from yeast and a variety of bacteria, this is the first report characterizing endopolyphosphatases that act on long chain inorganic polyphosphate (polyP). The activity from Saccharomyces cerevisiae, localized in vacuoles, has been purified to homogeneity from a strain that possesses vacuolar proteases. The endopolyphosphatase is a dimer of 35-kDa subunits. Distributive action on polyP750 produces shorter chains to a limit of about polyP60, as well as the more abundant release of polyP3; the Km for polyP750 is 185 nM. Endopolyphosphatases have been identified in a wide variety of sources, except for most eubacteria tested. The activity has been partially purified from rat and bovine brain where its abundance is about 10 times higher than in other tissues but less than 1/10 that of yeast; the limit product of digestion of the partially purified brain enzyme is polyP3.

Acid Anhydride Hydrolases↗