Syndecan-4 promotes the retention of phosphatidylinositol 4,5-bisphosphate in the plasma membrane

Although phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates syndecan-4 function, the potential influence of syndecan-4 on PIP₂ remains unknown. GFP containing PIP₂-binding-PH domain of phospholipase Cδ (GFP-PHδ) was used to monitor PIP₂. Syndecan-4 overexpression in COS-7 cells enhanced membrane translocation of GFP-PHδ, while the opposite was observed when syndecan-4 was knocked-down. PIP₂ levels were higher in total phospholipids extracted from rat embryo fibroblasts expressing syndecan-4. Syndecan-4-induced membrane targeting of GFP-PHδ was further enhanced by phosphoinositide-3-kinase inhibitor, but not by phospholipase C (PLC) inhibitor. Besides, both ionomycin and epidermal growth factor caused dissociation of GFP-PHδ from plasma membrane, an effect that was significantly delayed by syndecan-4 over-expression. Collectively, these data suggest that syndecan-4 promotes plasma membrane retention of PIP₂ by negatively regulating PLC-dependent PIP₂ degradation.     

Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.     

Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture

During the stationary phase of growth, after 7 to 12 d in culture, the levels of phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) decreased by 75% in plasma membranes of the red alga Galdieria sulphuraria. Concomitant with the decrease in PtdInsP(2) levels in plasma membranes, there was an increase in PtdInsP(2) in microsomes, suggesting that the levels of plasma membrane PtdInsP(2) are regulated differentially. The decline of PtdInsP(2) in plasma membranes was accompanied by a 70% decrease in the specific activity of PtdInsP kinase and by reduced levels of protein cross-reacting with antisera against a conserved PtdInsP kinase domain. Upon osmotic stimulation, the loss of PtdInsP(2)from the plasma membrane increased from 10% in 7-d-old cells to 60% in 12-d-old cells, although the levels of inositol 1,4,5-trisphosphate (InsP(3)) produced in whole cells were roughly equal at both times. When cells with low plasma membrane PtdInsP(2) levels were osmotically stimulated, a mild osmotic stress (12.5 mM KCl) activated PtdInsP kinase prior to InsP(3) production, whereas in cells with high plasma membrane PtdInsP(2), more severe stress (250 mM KCl) was required to induce an increase in PtdInsP kinase activity. The differential regulation of a plasma membrane signaling pool of PtdInsP(2) is discussed with regard to the implications for understanding the responsive state of cells.     

Regulation of cardiac IKs potassium current by membrane phosphatidylinositol 4,5-bisphosphate

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P2) was examined in guinea pig atrial myocytes using the whole-cell patch clamp method. IKs was elicited by depolarizing voltage steps given from a holding potential of -50 mV, and the effect of various test reagents on IKs was assessed by measuring the amplitude of tail current elicited upon return to the holding potential following a 2-s depolarization to +30 mV. Intracellular application of 50 microM wortmannin through a recording pipette evoked a progressive increase in IKs over a 10-15-min period to 208.5 +/- 14.6% (n = 9) of initial magnitude obtained shortly after rupture of the patch membrane. Intracellular application of anti-PtdIns(4,5)P2 monoclonal antibody also increased the amplitude of IKs to 198.4 +/- 19.9% (n = 5). In contrast, intracellular loading with exogenous PtdIns(4,5)P2 at 10 and 100 mum produced a marked decrease in the amplitude of IKs to 54.3 +/- 3.8% (n = 5) and 44.8 +/- 8.2% (n = 5), respectively. Intracellular application of neomycin (50 microM) or aluminum (50 microM) evoked an increase in the amplitude of IKs to 161.0 +/- 13.5% (n = 4) and 150.0 +/- 8.2% (n = 4), respectively. These results strongly suggest that IKs channel is inhibited by endogenous membrane PtdIns(4,5)P2 through the electrostatic interaction with the negatively charged head group on PtdIns(4,5)P2. Potentiation of IKs by P2Y receptor stimulation with 50 microM ATP was almost totally abolished when PtdIns(4,5)P2 was included in the pipette solution, suggesting that depletion of membrane PtdIns(4,5)P2 is involved in the potentiation of IKs by P2Y receptor stimulation. Thus, membrane PtdIns(4,5)P2 may act as an important physiological regulator of IKs in guinea pig atrial myocytes.     

Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86Rb+ uptake assay. We demonstrate direct modulation by PIP2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP2, KirBac1.1 is inhibited by PIP2 incorporated in the membrane (K(1/2) = 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP2 in the membrane.     

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells

Background

Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P₂ in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane_. For controlled and reversible depletion of PtdIns(4,5)P₂, voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.

Methods and Results

Expression and correct targeting of ECFP-PH-PLCδ1_, EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P₂ in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K⁺ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P₂ was identified.

Conclusions

Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.     

Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1] [2] [3] [4] [5] [6]. Recent studies have used the phospholipase C-delta1 (PLC-delta1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P(2) in vivo [7] [8] [9] [10]. Although these studies demonstrated that PI(4,5)P(2) is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-delta1 PH domain linked to enhanced green fluorescent protein (PH-EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell's dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH-EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH-EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P(2) spatially restricts actin polymerization and thereby affects cell spreading and retraction.     

Agonist-stimulated phosphatidylinositol 4,5-bisphosphate metabolism in the nervous system

Phosphatidylinositol 4,5-bisphosphate has recently gained prominence as the central component of a receptor transduction process which generates inositol 1,4,5-trisphosphate and diacylglycerol in stimulated cells. Both of these products of phospholipid metabolism have intracellular second messenger functions with diacylglycerol formation leading to activation of protein kinase C and inositol 1,4,5-trisphosphate stimulating Ca²⁺ release from intracellular stores in the endoplasmic reticulum. There is mounting evidence that the phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate is coupled to activated receptors by a guanylnucleotide binding protein, analogous to Ns and Ni which couple stimulatory and inhibitory hormone receptors to adenylate cyclase. Most of the key elements of this signalling mechanism have been found in the nervous system and so too has an entirely novel and unexpected inositol phosphate ester, inositol 1,3,4,5-tetrakisphosphate, whose function is not yet known. Phosphatidylinositol 4,5-bisphosphate breakdown, detected as the accumulation of inositol phosphates in agonist-stimulated nervous tissue preparations, is a functional response that has been useful in assessing the relevance of receptors identified by radioligand binding assays, and which provides an essential link between receptor occupation and responses such as neurotransmitter release and modulation of neuronal excitability.     

Adsorption of cations to phosphatidylinositol 4,5-bisphosphate

We investigated the binding of physiologically and pharmacologically relevant ions to the phosphoinositides by making 31P NMR, electrophoretic mobility, surface potential, and calcium activity measurements. We studied the binding of protons to phosphatidylinositol 4,5-bisphosphate (PIP2) by measuring the effect of pH on the chemical shifts of the 31P NMR signals from the two monoester phosphate groups of PIP2. We studied the binding of potassium, calcium, magnesium, spermine, and gentamicin ions to the phosphoinositides by measuring the effect of these cations on the electrophoretic mobility of multilamellar vesicles formed from mixtures of phosphatidylcholine (PC) and either phosphatidylinositol, phosphatidylinositol 4-phosphate, or PIP2; the adsorption of these cations depends on the surface potential of the membrane and can be described qualitatively by combining the Gouy-Chapman theory with Langmuir adsorption isotherms. Monovalent anionic phospholipids, such as phosphatidylserine and phosphatidylinositol, produce a negative electrostatic potential at the cytoplasmic surface of plasma membranes of erythrocytes, platelets, and other cells. When the electrostatic potential at the surface of a PC/PIP2 bilayer membrane is -30 mV and the aqueous phase contains 0.1 M KCl at pH 7.0, PIP2 binds about one hydrogen and one potassium ion and has a net charge of about -3. Our mobility, surface potential, and electrode measurements suggest that a negligible fraction of the PIP2 molecules in a cell bind calcium ions, but a significant fraction may bind magnesium and spermine ions.     

Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.

The metabolism of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat parotid acinar cells was investigated, particularly with regard to the effects of receptor-active agonists. Stimulation of cholinergic-muscarinic receptors with methacholine provoked a rapid disappearance of 40--50% of [32P]PtdIns(4,5)P2, but had no effect on PtdIns4P. Adrenaline, acting on alpha-adrenoceptors, and Substance P also stimulated net loss of PtdIns(4,5)P2. The beta-adrenoceptor agonist, isoprenaline, and the Ca2+ ionophore, ionomycin, failed to affect labelled PtdIns(4,5)P2 or PtdIns4P. By chelation of extracellular Ca2+ with excess EGTA, and by an experimental protocol that eliminates cellular Ca2+ release, it was demonstrated that the agonist-induced decrease in PtdIns(4,5)P2 is independent of both Ca2+ influx and Ca2+ release. These results may suggest that net PtdIns(4,5)P2 breakdown is an early event in the stimulus-response pathway of the parotid acinar cell and could be directly involved in the mechanism of agonist-induced Ca2+ release from the plasma membrane.     

Hair cells require phosphatidylinositol 4,5-bisphosphate for mechanical transduction and adaptation

After opening in response to mechanical stimuli, hair cell transduction channels adapt with fast and slow mechanisms that each depend on Ca(2+). We demonstrate here that transduction and adaptation require phosphatidylinositol 4,5-bisphosphate (PIP(2)) for normal kinetics. PIP(2) has a striking distribution in hair cells, being excluded from the basal region of hair bundles and apical surfaces of frog saccular hair cells. Localization of a phosphatidylinositol lipid phosphatase, Ptprq, to these PIP(2)-free domains suggests that Ptprq maintains low PIP(2) levels there. Depletion of PIP(2) by inhibition of phosphatidylinositol 4-kinase or sequestration by aminoglycosides reduces the rates of fast and slow adaptation. PIP(2) and other anionic phospholipids bind directly to the IQ domains of myosin-1c, the motor that mediates slow adaptation, permitting a strong interaction with membranes and likely regulating the motor's activity. PIP(2) depletion also causes a loss in transduction current. PIP(2) therefore plays an essential role in hair cell adaptation and transduction.     

Acanthamoeba myosin IC colocalizes with phosphatidylinositol 4,5-bisphosphate at the plasma membrane due to the high concentration of negative charge

The tail of Acanthamoeba myosin IC (AMIC) has a basic region (BR), which contains a putative pleckstrin homology (PH) domain, followed by two Gly/Pro/Ala (GPA)-rich regions separated by a Src homology 3 (SH3) domain. Cryoelectron microscopy had shown that the tail is folded back on itself at the junction of BR and GPA1, and nuclear magnetic resonance spectroscopy indicated that the SH3 domain may interact with the putative PH domain. The BR binds to acidic phospholipids, and the GPA region binds to F-actin. We now show that the folded tail does not affect the affinity of AMIC for acidic phospholipids. AMIC binds phosphatidylinositol 4,5-bisphosphate (PIP2) with high affinity (approximately 1 microm), but binding is not stereospecific. When normalized to net negative charge, AMIC binds with equal affinity to phosphatidylserine (PS) and PIP2. This and other data show that the putative PH domain of AMIC is not a typical PIP2-specific PH domain. We have identified a 13-residue sequence of basic-hydrophobic-basic amino acids within the putative PH domain that may be a major determinant of binding of AMIC to acidic phospholipids. Despite the lack of stereospecificity, AMIC binds 10 times more strongly to vesicles containing 5% PIP2 plus 25% PS than to vesicles containing only 25% PS, suggesting that AMIC may be targeted to PIP2-enriched regions of the plasma membrane. In agreement with this, AMIC colocalizes with PIP2 at dynamic, protrusive regions of the plasma membrane. We discuss the possibility that AMIC binding to PIP2 may initiate the formation of a multiprotein complex at the plasma membrane.     

Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate is a signaling phospholipid of the plasma membrane that has a dynamically changing concentration. In addition to being the precursor of inositol trisphosphate and diacylglycerol, it complexes with and regulates many cytoplasmic and membrane proteins. Recent work has characterized the regulation of a wide range of ion channels by phosphatidylinositol 4,5-bisphosphate, helping to redefine the role of this lipid in cells and in neurobiology. In most cases, phosphatidylinositol 4,5-bisphosphate increases channel activity, and its hydrolysis by phospholipase C reduces channel activity.     

Spermine increases phosphatidylinositol 4,5-bisphosphate content in permeabilized and nonpermeabilized HL60 cells

The polyamine spermine (N,N'bis[3-aminopropyl]-1,4-butanediamine) activates phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P5K) and phosphatidylinositol 4-kinase (PtdIns4K) in vitro. Spermine concentration increases that occur in proliferating cells were approximated in streptolysin O-permeabilized HL60 cells. When phospholipase C was activated by GTPgammaS in the presence of PITPalpha, 0.1-1.2 mM spermine evoked increases in PtdIns(4,5)P(2) contents in a dose-dependent manner to 110-170% of control and concomitantly decreased inositol phosphate formation by 10-50%. Spermine-induced increases in PtdIns(4,5)P(2) content in permeabilized cells also occurred during GTPgammaS stimulation in the absence of PITPalpha, were augmented in the presence of PITPalpha, occurred in unstimulated cells and were additive to PtdIns(4,5)P(2) formation evoked by ARF1, another activator of phosphoinositide kinases. Slowly developing spermine-evoked increases in PtdIns(4,5)P(2) contents occurred in nonpermeabilized cells that were abolished in the presence of a spermine transport inhibitor. Data are consistent with spermine at physiological concentrations evoking a PITPalpha-dependent shift in formation of PtdIns(4,5)P(2) from compartments that contained an active phospholipase C to compartments that were separated from an active PLC and from PtdIns(4,5)P(2) formed by ARF1.     

Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P(2). Disrupting Exo70-PI(4,5)P(2) interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P(2)-binding-deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P(2) interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.     

Oxidants depress the synthesis of phosphatidylinositol 4,5-bisphosphate in heart sarcolemma

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is the substrate for phosphoinositide-phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. PtdIns 4,5-P2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P2. Exposure to the xanthine-xanthine oxidase-ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine-xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an *OH scavenger) did not modify the H2O2-induced depression of the kinases, eliminating any role of *OH. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 microM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutathione induced a significant depression. Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the redox ratio was observed. Thiol modifiers (N-ethylmaleimide, methyl methanethiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction.     

Guanine nucleotide and pyrophosphate activate exogenous phosphatidylinositol 4,5-bisphosphate hydrolysis in rat liver plasma membranes

5'-guanylylimidodiphosphate (GppNHp) in the presence of deoxycholate, stimulated the phospholipase C-mediated hydrolysis of exogenous [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) to myo-[3H]inositol 1,4,5-trisphosphate in rat liver plasma membranes. Activation was not specific for guanine nucleotides as 5'-adenylylimidodiphosphate, imidodiphosphate and pyrophosphate stimulated the enzyme with similar efficacies and potencies. Enzyme activation by GppNHp was most pronounced when [3H]PIP2 was used as substrate. No added Ca++ was required for [3H]PIP2 breakdown but hydrolysis was inhibited by divalent ion chelators. GppNHp stimulation was apparent in the presence of Ca++ or Mg++ as well as chelator concentrations that partially inhibited the enzyme, indicating that this effect was not attributed to changes in affinity of these divalent cations for the enzyme or substrate. These results suggest that guanine nucleotides can stimulate the hydrolysis of exogenous [3H]PIP2 in rat liver membranes by a non-specific effect probably due to the interaction of the diphosphate moiety with the enzyme or substrate.     

Mechanism of ADP ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is required both as a substrate for the generation of lipid-derived second messengers as well as an intact lipid for many aspects of cell signaling, endo- and exocytosis, and reorganization of the cytoskeleton. ADP ribosylation factor (ARF) proteins regulate PI(4,5)P(2) synthesis, and here we have examined whether this is due to direct activation of Type I phosphatidylinositol 4-phosphate (PIP) 5-kinase or indirectly by phosphatidate (PA) derived from phospholipase D (PLD) in HL60 cells. ARF1 and ARF6 are both expressed in HL60 cells and can be depleted from the cells by permeabilization. Both ARFs increased the levels of PIP(2) (PI(4,5)P(2), PI(3,5)P(2), or PI(3,4)P(2) isomers) at the expense of PIP when added back to permeabilized cells. The PIP(2) could be hydrolyzed by phospholipase C, identifying it as PI(4,5)P(2). However, the ARF1-stimulated pool of PI(4,5)P(2) was accessible to the phospholipase C more efficiently in the presence of phosphatidylinositol transfer protein-alpha. To examine the role of PLD in the regulation of PI(4,5)P(2) synthesis, we used butanol to diminish the PLD-derived PA. PI(4,5)P(2) synthesis stimulated by ARF1 was not blocked by 0.5% butanol but could be blocked by 1.5% butanol. Although 0.5% butanol was optimal for maximal transphosphatidylation, PA production was still detectable. In contrast, 1.5% butanol was found to inhibit the activation of PLD by ARF1 and also decrease PIP levels by 50%. Thus the toxicity of 1.5% butanol prevented us from concluding whether PA was an important factor in raising PI(4,5)P(2) levels. To circumvent the use of alcohols, an ARF1 point mutant was identified (N52R-ARF1) that could selectively activate PIP 5-kinase alpha activity but not PLD activity. N52R-ARF1 was still able to increase PI(4,5)P(2) levels but at reduced efficiency. We therefore conclude that both PA derived from the PLD pathway and ARF proteins, by directly activating PIP 5-kinase, contribute to the regulation of PI(4,5)P(2) synthesis at the plasma membrane in HL60 cells.