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Multiple isomers of inositol pentakisphosphate in Epstein-Barr-virus- transformed (T5-1) B-lymphocytes. Identification of inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate.

ABSTRACT: Substantial amounts of three [3H]InsP5 isomers were detected in [3H]inositol-labelled human lymphoblastoid (T5-1) cells. Their structures were determined by h.p.l.c. [Phillippy & Bland (1988) Anal. Biochem. 175, 162-166], and by utilizing a stereospecific D-inositol 1,2,4,5,6-pentakisphosphate 3-kinase from Dictyostelium discoideum [Stephens & Irvine (1990) Nature (London) 346, 580-583]. The structures were: inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate. The relative proportions of these isomers (approx. 73:14:14 respectively) were unaffected by cross-linking anti-IgD receptors. The T5-1 cells also contained InsP6 and three Ins P4s, which were identified as the 1,3,4,5, 1,3,4,6 and 3,4,5,6 isomers. In incubations with permeabilized T5-1 cells, both 1,3,4,6 and 3,4,5,6 isomers of InsP4 were phosphorylated solely to Ins(1,3,4,5,6)P5. Permeabilized cells also dephosphorylated InsP6, even in the presence of a large excess of glucose 6-phosphate to saturate non-specific phosphatases. In the latter experiments the following isomers of InsP5 accumulated: D- and/or L-Ins(1,2,3,4,5)P5, plus D- and/or L-Ins(1,2,4,5,6)P5. This demonstration that multiple isomers of InsP5 may be formed in vivo and in vitro by a transformed lymphocyte cell line adds a new level of complexity to the study of inositol polyphosphate metabolism and function.

SUBMITTER: McConnell FM

PROVIDER: S-EPMC1130549 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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scyllo-inositol pentakisphosphate as an analogue of myo-inositol 1,3,4,5,6-pentakisphosphate: chemical synthesis, physicochemistry and biological applications.

Project description:myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.

| S-EPMC1892220 | biostudies-literature

Inositol phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphosphate 2-kinase and its role in substrate specificity.

Project description:Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1-Å crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110-140 within the N-lobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4-, 5-, and 6-positions are recognized initially by the C-lobe with subsequent interaction of the 1-position phosphate by Arg130 that stabilizes this residue and the N-lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N- and C-lobes and kinase activation.

| S-EPMC3403471 | biostudies-literature

Conformational stability of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) dictates its substrate selectivity.

Project description:Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

| S-EPMC3873539 | biostudies-literature

Roles of phosphate recognition in inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) substrate binding and activation.

Project description:Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.

| S-EPMC3772240 | biostudies-literature

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

Project description: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)P5)]. 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)P5, 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)P5 forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe3+ is similar to that of InsP6 and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P5 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)P5 also forms the polymetallic complexes [M4(H2L)] [where L is fully deprotonated Ins(1,3,4,5,6)P5]. However, unlike InsP6, Ins(1,3,4,5,6)P5 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)] x 23H2O or [Ca4(H2L)] x 16H2O whenever they exceed 135 and 56 microM 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)P5 in straightforward plots.

| S-EPMC2745655 | biostudies-literature

A novel inhibitor of the PI3K/Akt pathway based on the structure of inositol 1,3,4,5,6-pentakisphosphate.

Project description:BackgroundOwing to its role in cancer, the phosphoinositide 3-kinase (PI3K)/Akt pathway is an attractive target for therapeutic intervention. We previously reported that the inhibition of Akt by inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) results in anti-tumour properties. To further develop this compound we modified its structure to obtain more potent inhibitors of the PI3K/Akt pathway.MethodsCell proliferation/survival was determined by cell counting, sulphorhodamine or acridine orange/ethidium bromide assay; Akt activation was determined by western blot analysis. In vivo effect of compounds was tested on PC3 xenografts, whereas in vitro activity on kinases was determined by SelectScreen Kinase Profiling Service.ResultsThe derivative 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP(5)) is active towards cancer types resistant to InsP(5) in vitro and in vivo. 2-O-Bn-InsP(5) possesses higher pro-apoptotic activity than InsP(5) in sensitive cells and enhances the effect of anti-cancer compounds. 2-O-Bn-InsP(5) specifically inhibits 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro (IC(50) in the low nanomolar range) and the PDK1-dependent phosphorylation of Akt in cell lines and excised tumours. It is interesting to note that 2-O-Bn-InsP(5) also inhibits the mammalian target of rapamycin (mTOR) in vitro.ConclusionsInsP(5) and 2-O-Bn-InsP(5) may represent lead compounds to develop novel inhibitors of the PI3K/Akt pathway (including potential dual PDK1/mTOR inhibitors) and novel potential anti-cancer drugs.

| S-EPMC2813745 | biostudies-literature

A non-catalytic role for inositol 1,3,4,5,6-pentakisphosphate 2-kinase in the synthesis of ribosomal RNA.

Project description:Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.

| S-EPMC3613177 | biostudies-literature

STAT3 imparts BRCAness by impairing homologous recombination repair in Epstein-Barr virus-transformed B lymphocytes.

Project description:Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV re-shapes cellular DNA repair, ii) provide the first genome-wide evidence for insertions resulting from MMEJ in human cells, and iii) expand the range of cancers (EBV-related and -unrelated) that are likely to respond to synthetic lethal inhibitors given the high prevalence of cancers with constitutively active STAT3.

| S-EPMC7529304 | biostudies-literature

Inositol pentakisphosphate isomers bind PH domains with varying specificity and inhibit phosphoinositide interactions.

Project description:BackgroundPH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers.ResultsIn work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does.ConclusionsThese results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathways.

| S-EPMC3042905 | biostudies-literature

Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1).

Project description:The metabolic pathway(s) by which plants synthesize InsP6 (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49-67], while the identities of the genes that encode enzymes catalysing individual steps in these pathways are, with the notable exception of myo-inositol phosphate synthase and ZmIpk [Shi, Wang, Wu, Hazebroek, Meeley and Ertl (2003) Plant Physiol. 131, 507-515], unidentified. A yeast enzyme, ScIPK1, catalyses the synthesis of InsP6 by 2-phosphorylation of Ins(1,3,4,5,6)P5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, HsIPK1, is able to substitute for yeast ScIPK1, restoring InsP6 production in a Saccharomyces cerevisiae mutant strain lacking the ScIPK1 open reading frame (ScIpk1Delta). We have identified an Arabidopsis genomic sequence, AtIPK1, encoding an Ins(1,3,4,5,6)P5 2-kinase. Inclusion of the AtIPK1 protein in alignments of amino acid sequences reveals that human and Arabidopis kinases are more similar to each other than to the S. cerevisiae enzyme, and further identifies an additional motif. Recombinant AtIPK1 protein expressed in Escherichia coli catalysed the synthesis of InsP6 from Ins(1,3,4,5,6)P5. The enzyme obeyed Michaelis-Menten kinetics with an apparent V(max) of 35 nmol x min(-1) x (mg of protein)(-1) and a K(m) for Ins(1,3,4,5,6)P5 of 22 microM at 0.4 mM ATP. RT (reverse transcriptase)-PCR analysis of AtIPK1 transcripts revealed that AtIPK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of AtIPK1 in male and female organs of flower buds. Expression of AtIPK1 protein in an ScIpk1Delta mutant strain restored InsP6 production and rescued the temperature-sensitive growth phenotype of the yeast.

| S-EPMC1386007 | biostudies-other

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