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At least 865 records · Page 48Linked to original sources

PubMed1987

Alterations in phospholipid metabolism in the globally ischemic rat heart: emphasis on phosphoinositide specific phospholipase C activity.

The effect of global ischemia on myocardial ventricular membrane phospholipids was evaluated using a modified Langendorff preparation. Isolated rat hearts were perfused at 37 degrees C with oxygenated Krebs Ringer solution or rendered ischemic by cessation of perfusion (10 min to 3 h). Longer periods of ischemia were assessed by incubating preperfused (10 min) intact hearts in non-oxygenated Krebs (37 degrees C) for 6 to 18 h. Ischemia-induced alterations in phosphatidylinositol levels and phosphoinositide-specific phospholipase C (PI PLC) activity were assessed in detail, since inositol phospholipids and PI-PLC play putative roles in the regulation of cell function and Ca2+ homeostasis. Decreases in major membrane phospholipids (phosphatidylcholine, phosphatidylserine, cardiolipin and sphingomyelin) were demonstrated after long ischemic periods (6 to 18 h). While periods of ischemia (3 h or less) induced no change in structural phospholipids, an elevation in lysophosphatidylcholine and free fatty acids was found by 1 h. Notably a significant increase in phosphatidylinositol content and an accompanying decrease in cytosolic PI PLC activity was detected by 30 mins of ischemia. Reduced enzymic activity was not due to altered in vitro activation or deactivation of PI-PLC, to a change in the Ca2+ requirement of the enzyme, or to translocation of the enzyme from the cytosol to a membrane fraction. The isolated rat heart made globally ischemic for 30 mins under conditions described for this investigation shows signs of irreversible injury i.e. increased cell Ca2+ content and inability to initiate and maintain rhythmic contraction upon reperfusion. Therefore, it is possible that altered phosphoinositide metabolism may contribute to the evolution of ischemia-elicited irreversible cell injury.

Animals↗

PubMed1999

2-Chloroadenosine reverses hyperglycemia-induced inhibition of phosphoinositide synthesis in cultured human retinal pigment epithelial cells and prevents reduced nerve conduction velocity in diabetic rats.

The effect of the adenosine (AD) analog 2-chloroadenosine (C-AD) on glucose-induced inhibition of phosphoinositide synthesis was studied in human retinal pigment epithelial (RPE) cells by monitoring the level of the phosphatidylinositol (PI) synthase substrate, cytidine diphosphate diglyceride (CDP-DG). In high-aldose reductase (AR)-expressing RPE 91 cells, C-AD decreased CDP-DG at 5 mmol/L glucose and reversed the increase by 20 mmol/L glucose. AD deaminase (ADA), which inactivates endogenously released AD, potentiated the hyperglycemia-induced increase in CDP-DG. Theophylline, an AD-A1 and AD-A2 receptor antagonist, caused an increase in CDP-DG at 20 mmol/L glucose. C-AD did not alter CDP-DG in low-AR-expressing RPE 45 cells, but did decrease CDP-DG after cells were conditioned in 300 mmol/L glucose for 1 week (which induces AR). The mechanism by which AD regulates PI synthase in cells with high AR activity is unknown, but it is independent of Gi or Gs proteins, adenylate cyclase and phospholipase C (PLC) activation, myo-inositol (MI) uptake, or MI efflux. Administration of C-AD to streptozotocin-induced diabetic rats prevented the slowing of motor nerve conduction velocity (MNCV). Thus, AD derivatives, which reverse a glucose-induced deficit in phosphoinositide metabolism, might serve as a useful pharmacological tool to intervene in hyperglycemia-induced diabetic complications.

2-Chloroadenosine↗

PubMed2001

SKF83959 exhibits biochemical agonism by stimulating [(35)S]GTP gamma S binding and phosphoinositide hydrolysis in rat and monkey brain.

SKF83959, a benzazepine with high affinity for aminergic receptors, elicits behaviors such as grooming and vacuous chewing that are characteristic of dopamine D(1)-like receptor stimulation in rodents. Unlike classical D(1) agonists, however, SKF83959 does not stimulate adenylyl cyclase. Knowing that some D(1)-like receptors are coupled to phospholipase C-mediated signaling cascades in the brain, the present study aimed to determine whether SKF83959 exhibits an agonistic action at the biochemical level and also whether this benzazepine can modulate phosphoinositide hydrolysis in a manner that would be consistent with the behavioral effects of the drug. Similar to dopamine and the selective D(1)-like agonist SKF38393, SKF83959 competitively displaced the receptor binding of [(3)H]dopamine in an agonist-like manner, significantly stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate binding, and potently enhanced phospholipase C-mediated phosphoinositide hydrolysis in rat and monkey brain tissues. SKF83959 was generally more potent than SKF38393, whereas SKF38393 consistently exhibited greater pharmacological efficacy. These findings may implicate a role for the phospholipase C signaling cascade in the agonistic behavioral and antiparkinsonian activity of SKF83959. Dopamine-sensitive phospholipase C signaling should probably be considered in subsequent formulations of mechanisms and models of dopaminergic function in the normal or diseased brain.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1999

Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association.

The PTEN tumor suppressor is mutated in diverse human cancers and in hereditary cancer predisposition syndromes. PTEN is a phosphatase that can act on both polypeptide and phosphoinositide substrates in vitro. The PTEN structure reveals a phosphatase domain that is similar to protein phosphatases but has an enlarged active site important for the accommodation of the phosphoinositide substrate. The structure also reveals that PTEN has a C2 domain. The PTEN C2 domain binds phospholipid membranes in vitro, and mutation of basic residues that could mediate this reduces PTEN's membrane affinity and its ability to suppress the growth of glioblastoma tumor cells. The phosphatase and C2 domains associate across an extensive interface, suggesting that the C2 domain may serve to productively position the catalytic domain on the membrane.

Amino Acid Sequence↗

PubMed2001

A novel all helix fold of the AP180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis.

Clathrin-mediated endocytosis plays a major role in retrieving synaptic vesicles from the plasma membrane following exocytosis. This endocytic process requires AP180 (or a homolog), which promotes the assembly and restricts the size of clathrin-coated vesicles. The highly conserved 33 kDa amino-terminal domain of AP180 plays a critical role in binding to phosphoinositides and in regulating the clathrin assembly activity of AP180. The crystal structure of the amino-terminal domain reported herein reveals a novel fold consisting of a large double layer of sheets of ten alpha helices and a unique site for binding phosphoinositides. The finding that the clathrin-box motif is mostly buried and lies in a helix indicates a different site and mechanism for binding of the domain to clathrins than previously assumed.

Adaptor Proteins, Vesicular Transport↗

PubMed1997

The role of the phosphoinositide signalling system in the pathogenesis of sporadic Alzheimer's disease: a hypothesis.

Great advances have been made in recent years in our knowledge of the genetic mutations found in early onset familial Alzheimer's disease (AD) and their pathological consequences. The pathogenesis of sporadic AD, on the other hand, is less clear, although a central role of oxidative stress is indicated. In the AD brain, severe dysfunctions in the phosphoinositide signalling pathway have been reported. In view of the fact that (a) oxidative stress can adversely affect phosphoinositide breakdown and hence diacylglycerol-mediated activation of protein kinase C and (b) protein kinase C activation reduces the production of beta-amyloid peptide from amyloid precursor protein, it is possible that this represents a pathogenic pathway whereby oxidative stress can lead to amyloid deposition and the development of the disease.

Alzheimer Disease↗

PubMed2001

Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo.

Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)-DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA-DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.

Androstadienes↗

PubMed2001

Histamine N-methyltransferase regulates histamine-induced phosphoinositide hydrolysis in guinea pig cerebellum.

We report here that the dose-response curve of the histamine-stimulated phosphoinositide hydrolysis in the guinea pig cerebellar slices was shifted to the left when the slices were pretreated with SKF 91488 (100 microM), a specific inhibitor of histamine N-methyltransferase (HMT). In contrast, the pretreatment of the cerebellar slices with aminoguanidine (100 microM - 1 mM), an inhibitor of diamine oxidase, had no effect on histamine-induced phosphoinositide hydrolysis. HMT mRNA was expressed abundantly in cerebellum, especially in Purkinje cells. These observations suggest that HMT regulates histaminergic neurotransmission in guinea pig cerebellum more predominantly than diamine oxidase in histamine degradation.

Animals↗

PubMed1999

Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland.

The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4-9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) which reflects PI cycle activation. AVT (10(-5) and 10(-4) M) induced a significant increase in 32P labelling of PI, PIP and PIP2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V1 receptor antagonist. The desmopressin and AVP 4-9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 microg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

Animals↗

PubMed2001

Dictyostelium discoideum: a genetic model system for the study of professional phagocytes. Profilin, phosphoinositides and the lmp gene family in Dictyostelium.

Profilin is a key regulator of actin polymerization, and plays a pivotal role at the interface of the phosphoinositide signal transduction pathway and the cytoskeleton. Recent evidence suggests the involvement of profilin in the regulation of phagocytosis and macropinocytosis, and the transport along the endosomal pathway. Disruption of profilin leads to a complex phenotype that includes abnormal cytokinesis, a block in development and defects in the endosomal pathway. Macropinocytosis, fluid phase efflux and secretion of lysosomal enzymes were reduced, whereas the rate of phagocytosis was increased as compared to wild-type cells. The lmpA gene, a homolog of the CD36/LIMPII family, was identified as a suppressor for most of the profilin-minus defects. This gene encodes an integral membrane protein, it localizes to lysosomes and macropinosomes, and binds to phosphoinositides. Even though phosphatidylinositol lipids constitute only a small fraction of total lipids in the membranes of eukaryotic cells, they play an important role in vesicle transport, signal transduction and cytoskeletal regulation. Disruption of lmpA in wild-type cells resulted in defects in fluid phase efflux and macropinocytosis, but not in phagocytosis. The discovery and initial characterization of two additional members of the CD36/LIMPII family in Dictyostelium, lmpB and lmpC, that exhibit intriguing differences in developmental regulation and their putative sorting signals, suggests that a set of lysosomal integral membrane proteins contribute to the crosstalk between vesicles and cytoskeletal proteins.

Animals↗

PubMed2000

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis.

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.

Animals↗

PubMed2001

PTEN and myotubularin phosphoinositide phosphatases: bringing bioinformatics to the lab bench.

Phosphoinositides play an integral role in a diverse array of cellular signaling processes. Although considerable effort has been directed toward characterizing the kinases that produce inositol lipid second messengers, the study of phosphatases that oppose these kinases remains limited. Current research is focused on the identification of novel lipid phosphatases such as PTEN and myotubularin, their physiologic substrates, signaling pathways and links to human diseases. The use of bioinformatics in conjunction with genetic analyses in model organisms will be essential in elucidating the roles of these enzymes in regulating phosphoinositide-mediated cellular signaling.

Amino Acid Sequence↗

PubMed1997

Phosphoinositides as spatial regulators of membrane traffic.

Inositol phospholipids are a focus of renewed interest with the discovery of their unanticipated pivotal roles in membrane trafficking events. Reversible phosphorylation of phosphatidylinositol generates spatially localized signals on membranes that recruit or activate proteins essential for cell membrane budding, fission and fusion. Recent advances have taken place in the characterization of lipid kinases and phosphoinositide-regulated effector proteins, and in the elucidation of phospholipase D mediated mechanisms involving ADP ribosylation factor and Rho family proteins. The roles played by phosphoinositides in aspects of secretory granule formation, fusion and endocytosis indicate the importance of phosphorylated lipids for neurotransmitter release.

Animals↗

PubMed2002

Activation of phosphoinositide 3-kinase gamma by Ras.

BACKGROUND: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation. RESULTS: We show that posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3Kgamma could we detect any evidence of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate. CONCLUSIONS: Our data suggest that Ras activate PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.

Animals↗

PubMed2002

PTEN and myotubularin phosphatases: from 3-phosphoinositide dephosphorylation to disease.

The phosphatase and tensin homolog deleted on chromosome ten (PTEN) and myotubularin (MTM1) represent subfamilies of protein tyrosine phosphatases whose principal physiological substrates are D3-phosphorylated inositol phospholipids. As lipid phosphatases, PTEN- and MTM1-related (MTMR) proteins dephosphorylate the products of phosphoinositide 3-kinases and antagonize downstream effectors that utilize 3-phosphoinositides as ligands for protein targeting domains or allosteric activation. Here, we describe the cellular mechanisms of PTEN and MTMR function and their role in the etiology of cancer and other human diseases.

Disease↗

PubMed1997

Cloning and mutagenesis of the p110 alpha subunit of human phosphoinositide 3'-hydroxykinase.

Activation of phosphoinositide 3'-hydroxykinase (P13K) is required for mitogenic signal transduction by several growth factors and oncogenes. P13K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110 alpha catalytic subunit of human P13K. This clone is highly homologous (> 99% amino acid identity) to bovine brain p110 alpha, but contains 10 amino acid differences from the human p110 alpha sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp915, (Asp933 + Phe934)) yielded P13K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110 alpha protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c-fos, an immediate early gene product. In contrast, the catalytically impaired p110 alpha mutants as well as the p85 alpha subunit of P13K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by P13K is dependent upon the enzymatic activity of the p110 alpha subunit of P13K.

Amino Acid Sequence↗

PubMed1998

Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I.

BACKGROUND: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyphosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides. RESULTS: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P2 but not with Ins(1,4,5)P3. The acyl-modified PtdIns(4,5)P2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [3H]BZDC-Ins(1,4,5)P3-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix. CONCLUSIONS: The PtdIns(4,5)P2-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P2 and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.

Binding Sites↗

PubMed1997

Fc gammaRIIB1 inhibition of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization is integrated by CD19 dephosphorylation.

The B cell receptor for immunoglobulin G, Fc gammaRIIB1, is a potent transducer of signals that block antigen-induced B cell activation. Coligation of Fc gammaRIIB1 with B lymphocyte antigen receptors (BCR) causes premature termination of phosphoinositide hydrolysis and Ca2+ mobilization and inhibits proliferation. This inhibitory signal is mediated in part by phosphorylation of Fc gammaRIIB1 and recruitment of phosphatases; however, the molecular target(s) of effectors is unknown. Here we report that Fc gammaRIIB1 inhibition of BCR signaling is mediated in part by selective dephosphorylation of CD19, a BCR accessory molecule and coreceptor. CD19 dephosphorylation leads to failed CD19 association with phosphatidylinositol 3-kinase, and this in turn leads to termination of inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and Ca2+ influx. The results define a molecular circuit by which Fc gammaRIIB signals block phosphoinositide hydrolysis.

Animals↗