Chicoric Acid Presented NLRP3-Mediated Pyroptosis through Mitochondrial Damage by PDPK1 Ubiquitination in an Acute Lung Injury Model.

Chicoric acid (CA), a functional food ingredient, is a caffeic acid derivative that is mainly found in lettuce, pulsatilla, and other natural plants. However, the anti-inflammatory effects of CA in acute lung injury (ALI) remain poorly understood. This study was conducted to investigate potential drug usage of CA for ALI and the underlying molecular mechanisms of inflammation. C57BL/6 mice were given injections of liposaccharide (LPS) to establish the in vivo model. Meanwhile, BMDM cells were stimulated with LPS+ATP to build the in vitro model. CA significantly alleviated inflammation and oxidative stress in both the in vivo and in vitro models of ALI through the inhibition of NLR family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis. In addition, CA attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis in the in vivo and in vitro models of ALI by suppressing the production of reactive oxygen species (ROS) via inhibiting the Akt/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. CA inhibited the interaction between Akt at T308 and phosphoinositide-dependent kinase-1 (PDPK1) at S549, thus promoting the phosphorylation of the Akt protein. Furthermore, CA directly targeted the PDPK1 protein and accelerated PDPK1 ubiquitination, indicating that 91-GLY, 111-LYS, 126-TYR, 162-ALA, 205-ASP, and 223-ASP might be responsible for the interaction between PDPK1 and CA. In conclusion, CA from Lettuce alleviated NLRP3-mediated pyroptosis in the ALI model through ROS-induced mitochondrial damage by activating Akt/Nrf2 pathway via PDPK1 ubiquitination. The present study suggests that CA might be a potential therapeutic drug to treat or prevent ALI in pneumonia or COVID-19.

A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha.

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.

Epigallocatechin gallate (EGCG) inhibits type II phosphatidylinositol 4-kinases: a key component in pathways of phosphoinositide turnover.

Type II phosphatidylinositol (PtdIns) 4-kinases produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. Type II PtdIns 4-kinase alpha and beta have similar biochemical properties. To distinguish these isoforms Epigallocatechin gallate (EGCG) has been evaluated as a specific inhibitor. EGCG is the major active catechin in green tea having anti-inflammatory, antiatherogenic and cancer chemopreventive properties. The precise mechanism of actions and molecular targets of EGCG in early signaling cascades are not well understood. In the present study, we have shown that EGCG inhibits type II PtdIns 4-kinases (α and ß isoforms) and PtdIns 3-kinase activity in vitro. EGCG directly bind to both alpha and beta isoforms of type II PtdIns 4-kinases with a Kd of 2.62 µM and 1.02 µM, respectively. Type II PtdIns 4-kinase-EGCG complex have different binding pattern at its excited state. Both isoforms showed significant change in helicity upon binding with EGCG. EGCG modulates its effect by interacting with ATP binding pocket; the residues likely to be involved in EGCG binding were predicted by Autodock. Our findings suggest that EGCG inhibits two isoforms and could be a key to regulate T cell activation.

Misregulation of phosphoinositides in Arabidopsis thaliana decreases pollen hydration and maternal fertility.

Phosphoinositides are important lipids involved in membrane identity, vesicle trafficking, and intracellular signaling. In recent years, phosphoinositides have been shown to play a critical role in polarized secretion in plants, as perturbations of phosphoinositide metabolism, through loss of function mutants, result in defects in root hair elongation and pollen tube growth, where polarized secretion occurs rapidly. In the Brassicaceae, responses of stigmatic papillae to compatible pollen are also thought to involve highly regulated secretory events to facilitate pollen hydration and penetration of the pollen tube through the stigmatic surface. We therefore sought to analyze the female sporophyte fertility of the root hair defective4-1 mutant and the PI 4-kinase ß1/ß2 double mutant, which differentially affect phosphatidylinositol-4-phosphate (PI4P) levels. Stigmas from both mutants supported slower rates of pollen grain hydration, and the fecundity of these mutants was also diminished as a result of failed pollination events. This study therefore concludes that PI4P is integral to appropriate pistil responses to compatible pollen.

1,2,3,4,6-Penta-O-galloly-beta-D-glucose suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1α and signaling in LNCaP prostate cancer cells.

Hypoxia is the hallmark of solid tumors and contributes to tumor angiogenesis mainly through activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). In addition to upregulating vascular endothelial growth factor (VEGF) in angiogenesis, HIF-1 plays critical roles in the metabolism, proliferation, metastasis, and differentiation of cancer cells. We and others have previously shown that 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) from Oriental herbal medicine possesses anti-angiogenic, anti-tumorigenic, and anti-diabetic activities. In the present study, we report that PGG inhibits hypoxia-induced protein accumulation, transcriptional activation, and mRNA expression of HIF-1α in LNCaP prostate cancer cells. PGG reduced cellular and secreted VEGF levels as well as mRNA expression in LNCaP cells. PGG suppressed capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxia-induced LNCaP cells, indicating that PGG has anti-angiogenic activity under hypoxic condition. Furthermore, PGG reduced expression of phosphoinositide 3-kinase (PI3K) as well as phosphorylation of AKT and mammalian target of rapamycin (mTOR), but not extracellular signal-regulated kinase (ERK) in LNCaP cells under hypoxic condition. Consistently, LY294002, a specific PI3K inhibitor, enhanced the inactivation of HIF-1α and AKT by PGG in LNCaP cells. Taken together, our results demonstrate that PGG inhibits hypoxia-mediated accumulation of HIF-1α as well as its downstream signaling to VEGF and PI3K/AKT/mTOR pathway in LNCaP prostate cancer cells.

Tanshinone IIA induces mitochondria dependent apoptosis in prostate cancer cells in association with an inhibition of phosphoinositide 3-kinase/AKT pathway.

Tanshinone IIA (Tan IIA; 14,16-epoxy-20-nor-5(10),6,8,13,15-abietapentaene-11,12-dione), a phytochemical derived from the roots of Salvia miltiorrhiza BUNGE, has been reported to posses anti-angiogenic, anti-oxidant, anti-inflammatory and apoptotic activities. However, the cancer growth inhibitory/cytocidal effects and molecular mechanisms in prostate cancer cells have not been well studied. In the present study, we demonstrate that Tan IIA significantly decreased the viable cell number of LNCaP (phosphate and tensin homolog (PTEN) mutant, high AKT, wild type p53) prostate cancer cells more sensitively than against the PC-3 (PTEN null, high AKT, p53 null) prostate cancer cells. Tan IIA significantly increased TdT-mediated dUTP nick-end labeling (TUNEL) positive index and sub-G1 DNA contents of treated cells, consistent with apoptosis. Tan IIA treatment led to cleavage activation of pro-caspases-9 and 3, but not pro-caspase-8, and cleavage of poly (ADP ribose) polymerase (PARP), a caspase-3 substrate. Additionally, Tan IIA treatment induced cytochrome c release from the mitochondria into the cytosol and reduced mitochondrial membrane potential and suppressed the expression of mitochondria protective Bcl-2 family protein Mcl-1(L). Tan IIA reduced the expression of phosphoinositide 3-kinase (PI3K) p85 subunit, and the phosphorylation of AKT and mammalian target of rapamycin (mTOR) in a concentration-dependent manner. Moreover, the combination of Tan IIA and LY294002, a specific PI3K inhibitor, enhanced PARP cleavage of LNCaP and PC-3, but not in MDA-MB-231 breast cancer cells which do not contain detectable active AKT. The findings suggest that Tan IIA-induced apoptosis involves mitochondria intrinsic caspase activation cascade and an inhibition of the PI3K/AKT survival pathway.

Phosphoinositide 3-kinase/protein kinase B signaling pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis: complementarity with classical protein kinase C.

UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.

Functional cooperativity of enzymes of phosphoinositide conversion according to synergistic effects on pectin secretion in tobacco pollen tubes.

The Arabidopsis phosphoinositide kinases PI4Kß1 and PIP5K5 have been implicated in the control of directional vesicle trafficking underlying polar tip growth in pollen tubes. PI4Kß1 and PIP5K5 catalyze key consecutive steps of phosphoinositide conversion, and it appears obvious that phosphatidylinositol-4-phosphate formed by PI4Kß1 might act as a substrate for phosphatidylinositol-4,5-bisphosphate formation by PIP5K5. However, this hypothesis has not been experimentally addressed and distinct localization patterns of PI4Kß1, PIP5K5, and also PI-synthases (PIS) generating phosphatidylinositol suggest additional complexity. Here, the synergistic functionality of enzymes of phosphoinositide conversion was assessed. In tobacco and Arabidopsis pollen tubes, phosphoinositides influence the apical secretion of pectin, and increased pectin deposition results in characteristic morphological alterations. Catalytically active and dominant negative variants of PI4Kß1 and PIP5K5 were systematically co-expressed in tobacco pollen tubes and the incidence of morphologies related to enhanced pectin secretion was evaluated. The data support a proposed functional interplay of PI4Kß1 and PIP5K5 at the trans-Golgi network, mediating directional vesicle trafficking. Co-expression experiments additionally including PIS isoforms, PIS1 or PIS2, indicate that pectin secretion is synergistically mediated by PI4Kß1 and PIP5K5 acting on PtdIns formed by PIS2 rather than PIS1. Possible ramifications for the preferential channeling of phosphoinositide intermediates between particular isoforms of PI pathway enzymes are discussed.

Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach.

Phosphoinositide 3-kinases (PI3Ks) are among the most frequently activated signaling pathways in cancer. In chronic lymphocytic leukemia (CLL), signals from the microenvironment are critical for expansion of the malignant B cells, and cause constitutive activation of PI3Ks. CXCR4 is a key receptor for CLL cell migration and adhesion to marrow stromal cells (MSCs). Because of the importance of CXCR4 and PI3Ks for CLL-microenvironment cross-talk, we investigated the activity of novel, isoform-selective PI3K inhibitors that target different isoforms of the p110-kDa subunit. Inhibition with p110alpha inhibitors (PIK-90 and PI-103) resulted in a significant reduction of chemotaxis and actin polymerization to CXCL12 and reduced migration beneath MSC (pseudoemperipolesis). Western blot and reverse phase protein array analyses consistently demonstrated that PIK-90 and PI-103 inhibited phosphorylation of Akt and S6, whereas p110delta or p110beta/p110delta inhibitors were less effective. In suspension and MSC cocultures, PI-103 and PIK-90 were potent inducers of CLL cell apoptosis. Moreover, these p110alpha inhibitors enhanced the cytotoxicity of fludarabine and reversed the protective effect of MSC on fludarabine-induced apoptosis. Collectively, our data demonstrate that p110alpha inhibitors antagonize stromal cell-derived migration, survival, and drug-resistance signals and therefore provide a rational to explore the therapeutic activity of these promising agents in CLL.

The highly charged region of plant beta-type phosphatidylinositol 4-kinase is involved in membrane targeting and phospholipid binding.

In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The alpha-type PI4Ks (approximately 220 kDa) contain a PH domain, which is lacking in beta-type PI4Ks (approximately 120 kDa). Beta-type PI4Ks, exemplified by Arabidopsis AtPI4Kbeta and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant beta-type PI4Ks at present. The PPC region has a length of approximately 300 amino acids and harboring 11 (AtPI4Kbeta) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based "Sequence of Membrane-Targeting Detection" system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1-8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kbeta was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P2 in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant beta-type PI4Ks.

Tea polyphenol epigallocatechin 3-gallate impedes the anti-apoptotic effects of low-grade repetitive stress through inhibition of Akt and NFkappaB survival pathways.

V79 Chinese Hamster lung fibroblasts were subjected to repetitive low-grade stress through multiple exposures to 30 microM H2O2 in culture for 4 weeks. Akt/protein kinase B became phosphorylated at serine473 and threonine308 during this period of repetitive stress. Concurrent exposure of the cells to LY294002 (5 microM), a phosphoinositide-3 kinase inhibitor or 4.5 microM epigallocatechin 3-gallate (EGCG), a tea polyphenol almost completely blocked Akt activation by repetitive stress. Phosphorylation of I kappa B kinase (IKK) and transcriptional activity driven by nuclear factor kappa B (NFkappaB) were significantly enhanced by repetitive oxidative stress. These increases were largely abolished by simultaneous exposure to EGCG. The repetitively stressed cells demonstrated a significant resistance to apoptosis by subsequent acute stress in the form of ultraviolet radiation at 5 J/m2 or H2O2 (7.5 mM). The resistance to apoptosis conferred by repetitive stress was drastically reduced (>80%) by constant exposure to EGCG during the stress period while the presence of LY294002 or the NFkappaB inhibitor SN50 brought about a relatively moderate effect (about 50-65%). Our data indicate that activation of Akt and NFkappaB pro-survival pathways by repetitive low-grade stress results in a strong inhibition of the normal apoptotic response after subsequent acute stress. The tea polyphenol EGCG impedes the activation of both Akt and NFkappaB by repetitive stress and as a result preserves the normal apoptotic response during subsequent acute stress.

Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro.

A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses.

NCS-1 inhibits insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes through a phosphatidylinositol 4-kinase-dependent pathway.

Expression of NCS-1 (neuronal calcium sensor-1, also termed frequenin) in 3T3L1 adipocytes strongly inhibited insulin-stimulated translocation of GLUT4 and insulin-responsive aminopeptidase. The effect of NCS-1 was specific for GLUT4 and the insulin-responsive aminopeptidase translocation as there was no effect on the trafficking of the cation-independent mannose 6-phosphate receptor or the GLUT1 glucose transporter isoform. Moreover, NCS-1 showed partial colocalization with GLUT4-EGFP in the perinuclear region. The inhibitory action of NCS-1 was independent of calcium sequestration since neither treatment with ionomycin nor endothelin-1, both of which elevated the intracellular calcium concentration, restored insulin-stimulated GLUT4 translocation. Furthermore, NCS-1 did not alter the insulin-stimulated protein kinase B (PKB/Akt) phosphorylation or the recruitment of Cbl to the plasma membrane. In contrast, expression of the NCS-1 effector phosphatidylinositol 4-kinase (PI 4-kinase) inhibited insulin-stimulated GLUT4 translocation, whereas co-transfection with an inactive PI 4-kinase mutant prevented the NCS-1-induced inhibition. These data demonstrate that PI 4-kinase functions to negatively regulate GLUT4 translocation through its interaction with NCS-1.

A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis.

The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. Here we show that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. Our results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis.

A plant 126-kDa phosphatidylinositol 4-kinase with a novel repeat structure. Cloning and functional expression in baculovirus-infected insect cells.

Phosphatidylinositol metabolism plays a central role in signaling pathways in animals and is also believed to be of importance in signal transduction in higher plants. We report here the molecular cloning of a cDNA encoding a previously unidentified 126-kDa phosphatidylinositol (PI) 4-kinase (AtPI4Kbeta) from the higher plant Arabidopsis thaliana. The novel protein possesses the conserved domains present in animal and yeast PI 4-kinases, namely a lipid kinase unique domain and a catalytic domain. An additional domain, approximately 300 amino acids long, containing a high percentage (46%) of charged amino acids is specific to this plant enzyme. Recombinant AtPI4Kbeta expressed in baculovirus-infected insect (Spodoptera frugiperda) cells phosphorylated phosphatidylinositol exclusively at the D4 position of the inositol ring. Recombinant protein was maximally activated by 0.6% Triton X-100 but was inhibited by adenosine with an IC50 of approximately 200 microM. Wortmannin at a concentration of 10 microM inhibited AtPI4Kbeta activity by approximately 90%. AtPI4Kbeta transcript levels were similar in all tissues analyzed. Light or treatment with hormones or salts did not change AtPI4Kbeta transcript levels to a great extent, indicating constitutive expression of the AtPI4Kbeta gene.

Effects of Alzheimer's disease-related beta amyloid protein fragments on enzymes metabolizing phosphoinositides in brain.

Phosphatidylinositol 4-kinase (PI 4-kinase) and phosphatidylinositol 4-phosphate kinase (PIP kinase) were assayed in membranes prepared from samples of human frontal cortex initially frozen at autopsy. PI 4-kinase activity was significantly lower in Alzheimer's disease patients relative to age-matched controls or patients with Parkinson's disease. PIP kinase was not different in Alzheimer's versus age-matched controls. The beta amyloid protein fragment 1-40 inhibited PI 4-kinase activity in assays of control human or rat cortical membranes. Fragments 1-28 and 25-35 could not mimic the effects of fragment 1-40 while a reverse peptide 40-1 was equipotent. The inhibition of PI 4-kinase by fragment 1-40 was competitive with substrate. The beta amyloid protein fragments had diverse effects on phosphoinositide-specific phospholipase C (PI-PLC) as assayed in rat cortical membranes. Low concentrations of fragment 1-40 stimulated, while high concentrations of 1-40 or 40-1 inhibited PI-PLC activity. Fragment 25-35 stimulated PI-PLC nearly 3-fold, while fragment 1-28 had only minor effects on the enzyme. The results suggest alterations in phosphoinositide metabolism in Alzheimer's disease which could affect signal transduction and/or cytoskeletal organization.

Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol.

Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.

Transient temporal relationship between 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated synthesis and hydrolysis of polyphosphoinositides: desensitization of phospholipase C and the inositol lipid kinases upon long-term treatment of ascites cells by exogenous OAG.

In ascites tumor cells, phosphoinositide metabolism can be activated by short-term treatment with exogenously added 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the membrane-permeable analog of diacylglycerides (DAG). Quiescent cells prelabeled with D-myo-2-[3H]inositol and then stimulated with OAG (20 micrograms/ml of medium) reveal transient increases in the liberation of inositol 1,4-bis- and inositol 1,4,5-trisphosphate with peaks at 30 min, and a sustained accumulation of inositol phosphate 30 min after stimulation. The labeling patterns of the corresponding inositol lipids show transient activity profiles for phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and a sustained high activity level for PtdIns 30 min after OAG treatment. These data demonstrate a temporal relationship between synthesis and phospholipase C (PLC)-induced hydrolysis of these lipids. Simultaneous labeling of the cellular inositol phospholipids with [1-14C]arachidonic acid reveals modest accumulations after OAG stimulation. The relative 3H radioactivity distribution between the lipids and their inositol metabolites show that about 10% of the polyphosphoinositide pools are metabolically active. Long-term culturing of the cells (> 24 h) under OAG supplementation produces significant reductions in the catalytic activities of PLC and the PtdIns and PtdIns(4)P-specific kinases which is paralleled by a reduced radioactive labeling of PtdIns(4)P and PtdIns(4,5)P2 under these conditions. These data suggest that diglycerides affect the phosphoinositide metabolism by controlling PLC and phosphoinositide kinase activities probably via modification of membrane properties, and by functioning as modulator of other events.

Attenuation of phosphoinositidase activity and phosphatidylinositol bisphosphate level of bovine retinal capillary pericytes in high glucose.

Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined. When glucose in growth medium was increased from 5- to 15- or 30 mM, PIase activity was attenuated to 82% or 55%, respectively. In contrast, when glucose (5-, 15-, 30 mM) was added to an enzyme extract from cells grown in the standard growth medium (5 mM glucose, 0.04 mM myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose. When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase. Phosphatidylinositol bisphosphate (PIP2). Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol. The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium. However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation. These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions. Further explanation of the role of the altered hydrolysis of PIP2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.