3-kinase Activity Research Articles

Serotonin induces an increase in D- myo-inositol (1,4,5)-trisphosphate 3-kinase activity in rat brainstem slices

Serotonin robustly potentiated the activity of the InsP 3 3-kinase in rat brainstem slices. This potentiation was mediated through activation of 5-HT 2 receptors since it was only retrieved with the selective 5-HT 2 agonist DOI but not with the 5-HT 1A agonist 8OHDPAT. The enhancement of the InsP 3 3-kinase activity by serotonin is positively modulated by pretreatment of the slices with the phosphatase inhibitor okadaic acid. Moreover, the specific CaMKII antagonists KN-62 and KN-93 dramatically reduced the serotonin-evoked increase in the InsP 3 3-kinase activity. It is thus concluded that InsP 3 3-kinase up-regulation occurs through activation of PLC-coupled serotoninergic receptors and requires the phosphorylation of the enzyme by the ubiquitous multimeric protein kinase CaMKII.

Activation of the Insulin-like Growth Factor Type 1 Receptor by Deletion of Amino Acids 870–905

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Δ870–905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Δ870–905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both α and β-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Δ870–905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Δ870–905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870–905 exert a negative effect on the tyrosine kinase domain of the β-subunit of the IGF-1 R.

An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release

Glutamate release from nerve terminals is the consequence of Ca2+-triggered fusion of small synaptic vesicles with the presynaptic plasma membrane. ATP dependence of neurotransmitter release has been suggested to be founded, in part, on phosphorylation steps preceding membrane fusion. Here we present evidence for an essential role of phosphatidylinositol phosphorylation in stimulated release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes). Specifically, we show that a phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity resides on nerve terminal-derived small synaptic vesicles (SSVs) and that inhibition of the PtdIns 4-kinase activity in intact synaptosomes leads to attenuation of the evoked release of glutamate. The attenuation of transmitter release is reversible and correlates with respective changes in intrasynaptosomal PtdIns 4-kinase activity. Because only the Ca2+-dependent release of glutamate is affected, regulation appears to be at the level of exocytosis. Taken together, our data imply a mandatory role for PtdIns 4-kinase and phosphoinositide products in the regulated exocytosis of SSV in mammalian nerve terminals.

A Novel Phosphatidylinositol-5-phosphate 4-Kinase (Phosphatidylinositol-phosphate Kinase IIγ) Is Phosphorylated in the Endoplasmic Reticulum in Response to Mitogenic Signals

Here, we identify a novel rat phosphatidylinositol-5-phosphate 4-kinase, phosphatidylinositol-phosphate kinase IIgamma (PIPKIIgamma). PIPKIIgamma comprises 420 amino acids with a molecular mass of 47,048 Da, showing greater homology to the type IIalpha and IIbeta isoforms (61.1 and 63.7% amino acid identities, respectively) of phosphatidylinositol-phosphate kinase than to the type I isoforms. It is predominantly expressed in kidney, with low expression in almost all other tissues. PIPKIIgamma was found to have phosphatidylinositol-5-phosphate 4-kinase activity as demonstrated in other type II kinases such as PIPKIIalpha. The PIPKIIgamma that is present endogenously in rat fibroblasts, PC12 cells, and rat whole brain lysate or that is exogenously overexpressed in COS-7 cells shows a doublet migrating pattern on SDS-polyacrylamide gel electrophoresis. Alkaline phosphatase treatment and metabolic labeling in [32P]orthophosphate experiments revealed that PIPKIIgamma is phosphorylated in vivo, resulting in a shift in its electrophoretic mobility. Phosphorylation is induced by treatment of mitogens such as serum and epidermal growth factor. Immunostaining experiments and subcellular fractionation revealed that PIPKIIgamma localizes dominantly in the endoplasmic reticulum (ER). Phosphorylation also occurs in the ER. Thus, PIPKIIgamma may have an important role in the synthesis of phosphatidylinositol bisphosphate in the ER.

A Phosphatidylinositol 4-Kinase Pleckstrin Homology Domain That Binds Phosphatidylinositol 4-Monophosphate

Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kalpha) and Arabidopsis (AtPI4Kalpha). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio.

A specific increased expression of insulin receptor substrate 2 in pancreatic beta-cell lines is involved in mediating serum-stimulated beta-cell growth.

Certain nutrients and growth factors can stimulate pancreatic beta-cell growth. However, the appropriate mitogenic signaling pathways in beta-cells have been relatively undefined. In this study, differential gene expression in NEDH rat insulinoma was compared with NEDH rat primary islet beta-cells. Differential mRNA display analysis revealed an elevated expression in insulinoma of VL30 transposons, S24 ribosomal protein, and cytochrome-C oxidaseVIIc that is typical for cells undergoing mitosis. A gene candidate approach revealed that mRNA levels of the oncogenes c-fos and c-jun were equivalently expressed in insulinoma and islet cells, as was the mRNA for the mitogenic signal transduction molecule insulin receptor substrate (IRS)-1. However, in contrast to that of IRS-1, IRS-2 gene expression was 60- to 70-fold higher in the insulinoma tissue compared with islets, which was reflected at the protein as well as the mRNA level. The specific elevated IRS-2 expression was a consistent observation across all rodent pancreatic beta-cell lines. To investigate whether IRS-2 was functional, serum-stimulated beta-cell proliferation was examined in isolated insulinoma cells. After a 48-h period of serum withdrawal, 24 h of serum refeeding rendered an 8- to 10-fold increase in [3H]thymidine incorporation into insulinoma cells. This serum-stimulated DNA synthesis was prevented by inhibitors of tyrosine protein kinase and phosphatidylinositol (PI) 3-kinase activities, as well as the activation of mitogen-activated protein (MAP) kinase and p70S6K. Examination of IRS-mediated signal transduction pathways indicated that after 10-15 min of serum refeeding, there was increased tyrosine phosphorylation of IRS-2 and pp60, and PI 3-kinase recruitment to IRS-2. Serum also increased the association of growth factor-bound protein 2/murine sons of sevenless 1 protein to a PI 3-kinase/IRS-2 protein complex. Moreover, serum also activated MAP-kinase (erk-1 and erk-2 isoforms) and 70 kD S6 kinase. Thus IRS-mediated signal transduction pathways are functional in pancreatic beta-cells. It is conceivable that IRS-2 expression in beta-cells contributes to maintaining the islet beta-cell population, complementary to observations in the IRS-2 knockout mouse in which beta-cell mass is markedly reduced.

Phosphatidylinositol 4-Phosphate Synthesis in Immunoisolated Caveolae-like Vesicles and Low Buoyant Density Non-caveolar Membranes

This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae-like vesicles, to produce PtdIns4P. The addition of recombinant phosphatidylinositol transfer protein increased PtdIns 4-kinase activity both in immunoisolated caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membrane rafts are the main site for cellular PtdIns4P production.

Identification of a Potential Effector Pathway for the Trimeric Go Protein Associated with Secretory Granules: Go STIMULATES A GRANULE-BOUND PHOSPHATIDYLINOSITOL 4-KINASE BY ACTIVATING RhoA IN CHROMAFFIN CELLS

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles, and its activation inhibits the ATP-dependent priming of exocytosis. By using permeabilized cells, we previously described that the control exerted by the granule-bound Go on exocytosis may be related to effects on the cortical actin network through a sequence possibly involving Rho. To provide further insight into the function of Rho in exocytosis, we focus here on its intracellular localization in chromaffin cells. By immunoreplica analysis, immunoprecipitation, and confocal immunofluorescence, we found that RhoA is specifically associated with the membrane of secretory chromaffin granules. Parallel subcellular fractionation experiments revealed the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kinase activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan was mimicked by GAP-43, an activator of the granule-associated Go, and specifically inhibited by antibodies against Galphao. In addition, Clostridium botulinum C3 exoenzyme completely blocked the activation of phosphatidylinositol 4-kinase by mastoparan. We propose that the control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent phosphatidylinositol 4-kinase associated with the membrane of secretory granules.

A Genetic Screen for Aminophospholipid Transport Mutants Identifies the Phosphatidylinositol 4-Kinase, Stt4p, as an Essential Component in Phosphatidylserine Metabolism

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway.

PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359-2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.

Phosphatidylinositol 3-Kinase Is Involved in the Induction of Macrophage Growth by Oxidized Low Density Lipoprotein

Early atherosclerotic lesions are characterized by the presence of cholesterol-rich, macrophage-derived foam cells. It has recently been shown that macrophage proliferation occurs during the development of early lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this induction of macrophage growth include potentiation of mitogenic signal transduction by a component of oxidized LDL following internalization and degradation, interaction with integral plasma membrane proteins coupled to signaling pathways, or direct or indirect activation of growth factor receptors on the cell surface (e.g. GM-CSF receptor) through an autocrine/paracrine mechanism. The present study was undertaken to characterize some of the early intracellular signaling events by which oxidized LDL mediates macrophage cell growth. Extensively oxidized LDL increased protein-tyrosine phosphorylation and caused a 2-fold increase in phosphatidylinositol (PI) 3-kinase activity in phorbol ester-pretreated THP-1 cells (a human monocyte-like cell line). Similar concentrations of native LDL had no effect. Oxidized LDL also stimulated growth of resident mouse peritoneal macrophages, and this effect was reduced by 40-50% in cells treated with PI 3-kinase inhibitors (100 nM wortmannin or 20 microM LY294002). These results suggest that PI 3-kinase mediates part of the mitogenic effect of oxidized LDL, but parallel pathways involving other receptors and signal transduction pathways are likely also involved.

Differential Regulation of Muscarinic Acetylcholine Receptor-sensitive Polyphosphoinositide Pools and Consequences for Signaling in Human Neuroblastoma Cells

In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.

Reconstitution system based on cytosol-depleted cells to study the regulation of phospholipases C and D.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membrane-associated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes--PLD1 and PLD2--have been identified, although their regulatory mechanisms are yet to be fully understood. To study the regulation of PLD, we established a reconstitution system that allows the study of the PLD enzymes in their native environment while enabling the cytosol to be manipulated. Cells are permeabilized with a bacterial cytolysin (streptolysin O), which produces lesions in the plasma membrane, resulting in the release of cytosolic proteins. With increasing permeabilization times, guanosine 5'-[gamma-thio]triphosphate and receptor-activated PLD activity diminishes. Once the conditions for the run-down of the response is established, cellular factors, such as cytosol and purified proteins, can be added to these cells to restore activity. In addition to examining PLD activity, this reconstitution system allows the study of potential cellular targets of PA, such as phosphatidylinositol 4-phosphate (PIP) 5-kinase activity by monitoring PIP2 synthesis, and also functional outputs, such as exocytosis.

Inositol 1,4,5-trisphosphate turnover enzymes--activities and subcellular distribution in hepatocarcinogenesis.

The metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in homogenates and sub-fractions from normal rat liver and premalignant liver nodules was investigated. The activities of 5-phosphatase, expressed as pmol converted substrate per minute and mg protein, were equal when using the two substrates, and did not differ between normal and nodular homogenates. Subcellular fractions were purified by sequential steps of differential centrifugation and density gradient fractionation procedures. The total phosphatase activity was found to be distributed between cytosol (15%) and membraneous fractions (75%), with most of the enzyme activity residing in the plasma membranes. A doubling of phosphatase specific activity was seen in the nodular low density membrane fraction, containing Golgi apparatus and endosomes, as compared with normal liver. Inositol 1,4,5-trisphosphate 3-kinase activity was found to be exclusively cytosolic. No difference in this enzyme was seen between the two tissue types studied. Vasopressin (0.2 or 2 microM) had no effect either on phosphatase or kinase activity. The compartmentalization of inositol polyphosphate 5-phosphatase activity presents a possible explanation of earlier findings that premalignant liver tissue was able to respond with inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate formation after agonist stimulation.

Simulations of the effects of inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase activities on Ca 2+ oscillations

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P 3) is responsible for Ca 2+ mobilization in response to external stimulation in many cell types. The latter phenomenon often occurs as repetitive Ca 2+ spikes. In this study, the effect of the two Ins-1,4,5-P 3 metabolizing enzymes (Ins-1,4,5-P 3 3-kinase and 5-phosphatase) on the temporal pattern of Ca 2+ oscillations has been investigated. On the basis of the well-documented Ins-1,4,5-P 3 3-kinase stimulation by the Ca 2+/calmodulin complex and of the experimentally-determined kinetic characteristics of these enzymes, we predict that 5-phosphatase primarily controls the levels of Ins-1,4,5-P 3 and, thereby, the occurrence and frequency of Ca 2+ oscillations. Consequently, the model reproduces the experimental observation performed in Chinese hamster ovary cells that 5-phosphatase overexpression has a much more pronounced effect on the pattern of Ca 2+ oscillations than 3-kinase overexpression. We also investigated, in more detail, under which conditions a similar effect could be observed in other cell types expressing various Ins-1,4,5-P 3 3-kinase activities.

Cross-talk between phospholipase C and phosphoinositide 3-kinase signalling pathways

1321N1 astrocytoma cells have proved a valuable model system in which to study interactions between two major PtdIns (4,5) P2-utilizing signaling pathways, since they possess receptor populations which elicit independent activation of PI 3-kinase and a G-protein-dependent PLC respectively. Activation of PLC down-regulates PI 3-kinase by at least two mechanisms involving inhibition of IRS-1-associated PI 3-kinase and acute activation of a PtdIns (3,4,5) P3 5-phosphatase. PKB, which is an important early PI 3-kinase-dependent component of insulin signalling pathways, is also down-regulated by PLC-coupled agonists. The activation of PKB by insulin appears to involve a novel PtdIns (3,4,5) P3-dependent protein kinase, which we have named PDK1. The molecular mechanisms underlying PtdIns (3,4,5) P3-stimulated phosphorylation and activation of PKB by PDK1 are currently under investigation.

STT4 Is an Essential Phosphatidylinositol 4-Kinase That Is a Target of Wortmannin in Saccharomyces cerevisiae

Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate.

Isolation and molecular cloning of wortmannin-sensitive bovine type III phosphatidylinositol 4-kinases.

Agonist-sensitive phosphoinositide pools are maintained by recently-identified wortmannin (WT)-sensitive phosphatidylinositol (PI) 4-kinase(s) (Nakanishi, S., Catt, K. J., and Balla, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5317-5321). Two loosely membrane-associated WT-sensitive type III PI 4-kinases were isolated from bovine adrenal cortex as [3H]WT-labeled 110- and 210-kDa proteins. Based on peptide sequences from the smaller enzyme, a 3. 9-kilobase pair (kb) cDNA with an open reading frame encoding a 90-kDa protein was isolated from a bovine brain cDNA library. Expression of this cDNA in COS-7 cells yielded a 110-kDa protein with WT-sensitive PI 4-kinase activity. Northern blot analysis of a human mRNA panel showed a single approximately 3.8-kb transcript. Peptide sequences obtained from the 210-kDa enzyme corresponded to those of a recently described rat 230-kDa PI 4-kinase. A 6.5-kb cDNA containing an open reading frame of 6129 nucleotides that encoded a 230-kDa protein, was isolated from brain cDNA. Northern blot analysis of human mRNA revealed a major 7.5-kb transcript. The molecular cloning of these novel WT-sensitive type III PI 4-kinases will allow detailed analysis of their signaling and other regulatory functions in mammalian cells.

Exon–Intron Structure of a 2.7-kb Transcript of the STM7 Gene with Phosphatidylinositol-4-phosphate 5-Kinase Activity

The STM7 gene encodes a novel phosphatidylinositol-4-phosphate 5-kinase (PtdInsP 5-kinase) that is subject to alternative splicing and developmental control. We have recently presented data indicating that several splice variants of STM7 incorporate elements of the X25 sequence, previously implicated in the pathogenesis of Friedreich's ataxia by the detection of an intronic GAA repeat expansion as the predominant mutation in affected individuals. We now report the exon–intron structure of STM7.I and primer sequences designed to facilitate full characterization, including details relating to a novel exon (STM7; exon 17) derived from the 3′-UTR of the PRKACG gene. The detection of a mutation(s) within these exons would provide additional support for the hypothesis that a defect in phosphoinositide metabolism gives rise to the disease phenotype.

Carbamoylcholine regulation of polyphosphoinositide synthesis and hydrolysis in cultured, dispersed, digitonin-permeabilized mouse pancreatic islet cells.

Continuing formation of inositol phosphates during stimulation of pancreatic beta-cells by hormones and neurotransmitters requires the continued synthesis of the polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2) from phosphatidylinositol (PI). In the present study we have investigated how this pathway and the activity of phosphoinositide-specific phospholipase C (PI-PLC) are regulated by carbamoylcholine (CCh), Ca2+, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), GTP gamma S and NaF in 44-h [3H]inositol-labelled, dispersed and digitonin-permeabilized mouse pancreatic islet cells. CCh stimulated not only PI-PLC (G-protein-mediated) but also, by an as yet unknown mechanism, significantly enhanced PI 4-kinase activity, estimated as the PIP:PI ratio, by 100%, and further increased the flux from PI to PIP and PIP2, GTP gamma S and NaF mimicked the effects of CCh on PI-PLC but had no effect on the levels of PIP and PIP2, TPA raised the PIP:PI ratio by 75%. In addition TPA counteracted the CCh stimulation of PI-PLC. There was no effect of 10(-6) mol/l Ca2+ on the levels of PIP and PIP2. Experiments with quinacrine and adenosine confirmed that PI-PLC and PI 4-kinase could be regulated independently of each other. In conclusion, these data point to differential regulation of polyphosphoinositide synthesis and breakdown.