Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet alpha-granule secretion

To better understand the molecular mechanisms of platelet granule secretion, we have evaluated the role of type II phosphatidylinositol (PtdIns) 5-phosphate 4-kinase in agonist-induced platelet alpha-granule secretion. SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets was inhibited by either antibodies directed at type II PtdIns 5-phosphate 4-kinase or by a kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIbeta PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated alpha-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated alpha-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIbeta PtdIns 5-phosphate 4-kinase. Immunoblot analysis demonstrated that type II PtdIns 5-phosphate 4-kinase remained associated with SL-O-permeabilized platelets when incubated in the presence, but not the absence, of SFLLRN. This SFLLRN-induced translocation of type II PtdIns 5-phosphate 4-kinase was blocked by either the protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. In addition to stimulating alpha-granule secretion, both SFLLRN and PMA enhanced the association of a fluorescein isothiocyanate-labeled peptide derived from the PtdIns (4,5)P(2)-binding domain of gelsolin to permeabilized platelets. Agonist-induced recruitment of the PtdIns (4,5)P(2)-binding domain was inhibited by neomycin, bisindolylmaleimide I, and anti-type II PtdIns 5-phosphate 4-kinase antibody. These results suggest a mechanism whereby protein kinase C-mediated translocation of type II PtdIns 5-phosphate 4-kinase leads to the recruitment of PtdIns (4,5)P(2)-binding proteins.     

Gibberellin signal in barley aleurone: early activation of PLC by G protein mediates amylase secretion

The early events related to intracellular GA-signals in aleurone are not clearly established. We demonstrate that GA treatment induced increases in phosphatidylinositol 4P, 5-kinase (PtdInsP-k), diacylglycerol kinase (DAG-k) and phosphatidate kinase (PA-k) activities in barley aleurone within 5 min. The response to GA was also observed as a rapid and transient InsPs/InsP ₃ time-dependent accumulation. U73122, a phospholipase C (PLC) inhibitor, reduced the InsPs and InsP ₃ levels and amylase secretion. The G protein activator Mas7 was able to trigger the -amylase secretion as strongly as GA did; U73122, also reduced this effect. ABA evoked only an increase in phosphatidic acid (PtdOH) and diacylglycerol pyrophosphate (DGPP) levels. This is the first time that a rapid and transient response to GA in correlation with amylase secretion, involving PLC and G protein as well as PA-k activity in the GA signalling pathway, has been demonstrated in aleurone cells.     

Analysis of the catalytic domain of phosphatidylinositol 4-kinase type II

Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KIIalpha, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme.     

Phosphatidylinositol 4,5-bisphosphate mediates Ca2+-induced platelet alpha-granule secretion: evidence for type II phosphatidylinositol 5-phosphate 4-kinase function

To understand the molecular basis of granule release from platelets, we examined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in alpha-granule secretion. Streptolysin O-permeabilized platelets synthesized PtdIns(4,5)P(2) when incubated in the presence of ATP. Incubation of streptolysin O-permeabilized platelets with phosphatidylinositol-specific phospholipase C reduced PtdIns(4,5)P(2) levels and resulted in a dose- and time-dependent inhibition of Ca(2+)-induced alpha-granule secretion. Exogenously added PtdIns(4,5)P(2) inhibited alpha-granule secretion, with 80% inhibition at 50 microm PtdIns(4,5)P(2). Nanomolar concentrations of wortmannin, 33.3 microm LY294002, and antibodies directed against PtdIns 3-kinase did not inhibit Ca(2+)-induced alpha-granule secretion, suggesting that PtdIns 3-kinase is not involved in alpha-granule secretion. However, micromolar concentrations of wortmannin inhibited both PtdIns(4,5)P(2) synthesis and alpha-granule secretion by approximately 50%. Antibodies directed against type II phosphatidylinositol-phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) also inhibited both PtdIns(4,5)P(2) synthesis and Ca(2+)-induced alpha-granule secretion by approximately 50%. These antibodies inhibited alpha-granule secretion only when added prior to ATP exposure and not when added following ATP exposure, prior to Ca(2+)-mediated triggering. The inhibitory effects of micromolar wortmannin and anti-type II phosphatidylinositol-phosphate kinase antibodies were additive. These results show that PtdIns(4,5)P(2) mediates platelet alpha-granule secretion and that PtdIns(4,5)P(2) synthesis required for Ca(2+)-induced alpha-granule secretion involves the type II phosphatidylinositol 5-phosphate 4-kinase-dependent pathway.     

Chromaffin granule-associated phosphatidylinositol 4-kinase activity is required for stimulated secretion.

Permeabilized bovine adrenal chromaffin cells have been used to characterize the MgATP requirement of processes preceding exocytosis. Incubation of primary cultures with the membrane-permeable phenylarsine oxide (PAO) at 20 microM inhibited the phosphorylation of phosphatidylinositol (PtdIns) and completely blocked secretion. This block could be reversed by addition of 2,3-dimercaptopropanol to the permeabilized cells. Simultaneous addition of [gamma32P]ATP and 2,3-dimercaptopropanol permitted a comparison between recovery of secretion and phosphorylation of intracellular components. Recovery of secretion closely correlated with phosphorylation of PtdIns and PtdIns4P. Subcellular fractionation of permeabilized cells after recovery of secretion revealed that the majority of newly phosphorylated PtdIns4P was localized on the chromaffin granules. In accordance with these results, PtdIns 4-kinase activity was found in protein extracts of permeabilized cells as well as associated with purified chromaffin granules, sensitive in both cases to PAO. Additionally, PtdIns 4-kinase activity in these two assays was inhibited by quercetin. In permeabilized cells, quercetin decreased the levels of labeled PtdIns4P and Ptdlns(4,5)P2 and inhibited secretion. Our data suggest that a chromaffin granule-associated PtdIns 4-kinase acts in the priming of exocytosis.     

Phosphoinositide 3-kinase: A new effector in signal transduction?

Interest in phosphopinositide 3-kinase (PI 3-kinase) has been fuelled by its identification as a major phosphotyrosyl protein detected in cells following growth factor stimulation and oncogenic transformation. It is found complexed with activated growth factor receptors and non-receptor tyrosine kinases, thus suggesting that it participates in the signal transduction pathways initiated by the activation of tyrosine kinases. PI 3-kinase phosphorylates the 3-position in the inositol ring of the well known inositol phospholipids in vitro giving phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [PtdIns3P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃], respectively. The cellular levels of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ rapidly increase in circumstances where PI 3-kinase becomes complexed with tyrosine kinases. Accumulation of the same lipids also occurs in platelets and neutrophils following stimulation of G-protein linked -thrombin and chemotactic peptide receptors, respectively, leading to speculation that one or both of these lipids is a new second messenger whose function is not yet known. This review brings together recent information on the isolation, characterization and regulation of PI 3-kinase, the cellular occurrence of 3-phosphorylated inositol phospholipids and possible functions of the PI 3-kinase pathway in cell signalling.     

Essential role of phosphoinositide 3-kinase in leptin-induced K(ATP) channel activation in the rat CRI-G1 insulinoma cell line

The mechanism by which leptin increases ATP-sensitive K(+) (K(ATP)) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of K(ATP) channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) mimicked the effect of leptin by increasing K(ATP) channel activity in whole-cell and inside-out current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P(3), from increasing K(ATP) channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and K(ATP) channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P(3) as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of K(ATP) channels in this cell line. Although PtdIns(3,4,5)P(3) appears a plausible and attractive candidate for the messenger that couples K(ATP) channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P(3) demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P(3). Possible mechanisms to explain the involvement of PI 3-kinases in K(ATP) channel regulation are discussed.     

Phosphatidylinositol 3- and 4-phosphate modulate actin filament reorganization in guard cells of day flower

Phosphatidylinositol 3-kinases (PtdIns 3-kinases) that produce phosphatidylinositol (3,4,5) triphosphate (PtdIns(3,4,5)P₃) are considered to be important regulators of actin dynamics in animal cells. In plants, neither PtdIns(3,4,5)P₃ nor the enzyme that produces this lipid has been reported. However, a PtdIns 3-kinase that produces phosphatidylinositol 3-phosphate (PtdIns3P) has been identified, suggesting that PtdIns3P, instead of PtdIns(3,4,5)P₃, regulates actin dynamics in plant cells. Phosphatidylinositol 4-kinase (PtdIns 4-kinase) is closely associated with the actin cytoskeleton in plant cells, suggesting a role for this lipid kinase and its product phosphatidylinositol 4-phosphate (PtdIns4P) in actin-related processes. Here, we investigated whether or not PtdIns3P or PtdIns4P plays a role in actin reorganization induced by a plant hormone abscisic acid (ABA) in guard cells of day flower ( Commelina communis ). ABA-induced changes in actin filaments were inhibited by LY294002 (LY) and wortmannin (WM), inhibitors of PtdIns3P and PtdIns4P synthesis. Expression of PtdIns3P- and PtdIns4P-binding domains also inhibited ABA-induced actin reorganization in a manner similar to LY and WM. These results suggest that PtdIns3P and PtdIns4P regulate actin dynamics in guard cells. Furthermore, we demonstrate that PtdIns3P exerts its effect on actin dynamics, at least in part, via generation of reactive oxygen species (ROS) in response to ABA.     

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.     

Phosphorylation of signaling phospholipids in Coffea arabica cells

Membrane preparations of Coffea arabica suspension cells were incubated in the presence of 〚³²P〛γ-ATP. After lipid extraction and separation by thin layer chromatography, the following phosphorylated lipids were detected: phosphatidylinositol 4,5 bis-phosphate (PtdIns4,5P₂), lyso-phosphatidylinositol 4-phosphate (LPtdIns4P), phosphatidylinositol 4-phosphate (PtdIns4P), diacylglycerol pyrophosphate (DGPP), lyso-phosphatidic acid (LPA) and phosphatidic acid (PA). This suggests the presence of phosphatidylinositol (EC 2.7.1.67), phosphatidylinositol 4 phosphate (EC 2.7.1.68), diacylglycerol (EC 2.7.1.107) and monoacylglycerol (EC 2.1.1.94) kinases. The activities of these lipid kinases changed during the culture period of the C. arabica cells reaching peak at day 7 of culture; however, enzymatic activities were very low before and after day 7. The behavior of these lipid kinases in the presence of their respective substrates and exogenous substrates such as ATP was characterized. The apparent K_m values for ATP of all the lipid kinase activities were lower than 30 μM. All kinase activities assayed were totally dependent on the presence of Mg²⁺ and were unable to use Mn²⁺ or Ca²⁺ which produced a strong inhibition of all the lipid kinase activities. By using polyclonal antibodies against PtdIns 4-kinase and PtdInsP 5-kinase, we were able to identify at least two putative isoforms for the PtdIns 4-kinase and one for the PtdInsP 5-kinase. In both cases, the correlation of the amount of these proteins with their respective kinase activities depended on the culture cycle. The present work describes for the first time the characterization of the lipid kinases of C. arabica suspension cells, and the correlation of these activities with the culture cycle.     

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.     

Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents

Neutrophil priming by agents such as TNF-alpha and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-alpha or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10-60 s), which paralleled the augmented superoxide anion (O2-) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2- release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2- generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2- generation, that PI3-kinase-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.     

A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix.

Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.     

Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses

Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3-phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mM H(2)O(2) caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3, 4)P(2)), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and PtdIns(3,4)P(2) levels. The increase in PtdIns(3,4)P(2) levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P(3) and PtdIns(3, 4)P(2). p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P(2) levels. Increased PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P(2) can accumulate independently of PtdIns(3,4, 5)P(3) and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P(3).     

Binding of elongation factor eEF1A2 to phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and phosphatidylinositol 4-phosphate generation

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.     

A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms

Several RNA viruses have recently been shown to hijack members of the host phosphatidylinositol (PtdIns) 4-kinase (PI4K) family of enzymes. They use PI4K to generate membranes enriched in phosphatidylinositide 4-phosphate (PtdIns4P or PI4P) lipids, which can be used as replication platforms. Viral replication machinery is assembled on these platforms as a supramolecular complex and PtdIns4P lipids regulate viral RNA synthesis. This article highlights these recent studies on the regulation of viral RNA synthesis by PtdIns4P lipids. It explores the potential mechanisms by which PtdIns4P lipids can contribute to viral replication and discusses the therapeutic potential of developing antiviral molecules that target host PI4Ks as a form of panviral therapy.