Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.     

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.     

牛小脑肌醇磷脂激酶PI(4)K高产率纯化与特征

对牛小脑膜区肌醇磷脂激酶进行了11 500倍纯化,过程包括：TritonX-100抽提,硫酸铵沉淀,阳离子交换层析(phosphocellulose),亲和层析(Heparin Sepharose CL-6B)和阴离子交换层析(DEAE10,FPLC)等.纯化程度可达95％以上,对SDS-PAGE电泳结果进行扫描分析测其分子质量为56 ku.纯化的肌醇磷脂激酶的特异活性为450 nmol/mg·min, 动力学性质表现为ATP的表观K_m值为7.9×10^-7 mol/L,PI的表观K_m值为6.6×10^-7 mol/L. 腺嘌呤核苷是该酶的有效抑制剂,3.5×10^-7 mol/L腺嘌呤核苷可使该酶活力降低约50％,而TritonX-100对该酶活力具有刺激作用,0.5% TritonX-100可使该酶表现为最高活力.     

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.     

Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase

In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (K(D) = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.     

Affinity labelling of the active site of brain phosphatidylinositol 4-kinase with 5'-fluorosulphonylbenzoyl-adenosine.

5'-p-Fluorosulphonylbenzoyl-adenosine (FSO2BzAdo), an affinity labelling analogue of ATP, was used to label the active site of sheep brain phosphatidylinositol 4-kinase (PtdIns 4-kinase). The incubation of PtdIns 4-kinase with concentrations of FSO2BzAdo as low as 50 microM resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50 microM FSO2BzAdo). The kinetics of inactivation of PtdIns 4-kinase by FSO2BzAdo suggest a two-step mechanism, in which a rapid reversible binding of FSO2BzAdo to the enzyme is followed by a covalent sulphonation step. The first-order rate constant (k2) for the inactivation of PtdIns 4-kinase was calculated to be 0.063 min-1, and the steady-state constant of inactivation (Ki) to be 200 microM. Preincubation of the enzyme with either ATP plus Mg2+, or PtdIns alone, prior to addition of FSO2BzAdo reduced the degree of inactivation of the enzyme; suggesting that FSO2BzAdo binds within the active site PtdIns 4-kinase. Moreover, since ATP plus Mg2+ provided the greatest protection against inactivation, it is concluded that the main site of labelling of PtdIns 4-kinase by FSO2BzAdo is within the ATP-binding site of the enzyme. Results obtained from chemical modification experiments, which employed pyridoxal 5'-phosphate and tetranitromethane, are consistent with a catalytically-essential lysine being present within the ATP-binding site of PtdIns 4-kinase. Therefore, it is hypothesised that the inactivation of PtdIns 4-kinase by FSO2BzAdo may be due to the labelling of this lysine residue.     

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.     

Separation and identification of two phosphatidylinositol 4-kinase activities in bovine uterus

Growth factor-activated second messenger pathways are mediated in part via breakdown products of phosphoinositides. We have separated two phosphatidylinositol (PtdIns) 4-Kinases from bovine uteri which appear to be regulated independently. The predominant type II enzyme previously was purified to apparent homogeneity; the type I enzyme has been purified approximately 1000 fold (specific activity, approximately 30 nmoles/mg/min). The type I and type II enzymes differ sharply in apparent Km for ATP and response to divalent cations. In contrast to type II enzyme, type I PtdIns kinase was resistant to inhibition by adenosine, inhibited by increasing concentrations of Triton X-100, and less stable to storage than type II enzyme at pH values below 6.5 and above 8.5. Type I PtdIns 4-kinase has an apparent molecular mass of approximately 200 kD and type II enzyme of approximately 80 kD. Using both enzymatic and chemical criteria, both enzymes specifically phosphorylated the fourth hydroxyl group of PtdIns. The results thus establish the presence of two distinct and separate enzymes catalyzing PtdIns 4-kinase activity with different physical, kinetic, and regulatory properties, suggesting an important site for the regulation of second messenger signals transducing the responsiveness of cells to growth factors.     

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.     

A phosphotransferase that generates phosphatidylinositol 4-phosphate (PtdIns-4-P) from phosphatidylinositol and lipid A in Rhizobium leguminosarum. A membrane-bound enzyme linking lipid a and ptdins-4-p biosynthesis

Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unusual phosphate-deficient lipid A found in this organism. The enzyme has been solubilized with Triton X-100 and purified 80-fold. As shown by co-purification and thermal inactivation studies, the 4'-phosphatase catalyzes not only the hydrolysis of (Kdo)2-[4'-32P]lipid IVA but also the transfer the 4'-phosphate of Kdo2-[4'-32P]lipid IVA to the inositol headgroup of phosphatidylinositol (PtdIns) to generate PtdIns-4-P. Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizobium meliloti, or the nodulation-defective mutant 24AR of R. leguminosarum. The specific activity for the phosphotransferase reaction is about 2 times higher than that of the 4'-phosphatase. The phosphotransferase assay conditions are similar to those used for PtdIns kinases, except that ATP and Mg2+ are omitted. The apparent Km for PtdIns is approximately 500 microM versus 20-100 microM for most PtdIns kinases, but the phosphotransferase specific activity in crude cell extracts is higher than that of most PtdIns kinases. The phosphotransferase is absolutely specific for the 4-position of PtdIns and is highly selective for PtdIns as the acceptor. The 4'-phosphatase/phosphotransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns. A phosphoenzyme intermediate may account for the dual function of this enzyme, since a single 32P-labeled protein species (Mr approximately 68,000) can be trapped and visualized by SDS gel electrophoresis of enzyme preparations incubated with Kdo2-[4'-32P]lipid IVA. Although PtdIns is not detected in cultures of R. leguminosarum/etli (CE3), PtdIns may be synthesized during nodulation or supplied by plant membranes, given that soybean PtdIns is an excellent phosphate acceptor. A bacterial enzyme for generating PtdIns-4-P and a direct link between lipid A and PtdIns-4-P biosynthesis have not been reported previously.     

Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a PtdIns 4-kinase from lymphocytes.

A PtdIns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a PtdIns 4-kinase from splenic lymphocytes. The enzyme activity was solubilized from spleen particulate fraction with Triton X-100 and chromatographed sequentially on phosphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturation of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein. Characterization of the enzyme showed that it was a type II PtdIns 4-kinase. Polyclonal antibodies raised against PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymphoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes with Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical chromatographic behaviour, molecular mass, immunoreactivity, K(m) values for PtdIns and inhibition by adenosine.     

Design of drug-resistant alleles of type-III phosphatidylinositol 4-kinases using mutagenesis and molecular modeling

Molecular modeling and site directed mutagenesis were used to analyze the structural features determining the unique inhibitor sensitivities of type-III phosphatidylinositol 4-kinase enzymes (PI4Ks). Mutation of a highly conserved Tyr residue that provides the bottom of the hydrophobic pocket for ATP yielded a PI4KIIIbeta enzyme that showed greatly reduced wortmannin sensitivity and was catalytically still active. Similar substitutions were not tolerated in the type-IIIalpha enzyme rendering it catalytically inactive. Two conserved Cys residues located in the active site of PI4KIIIalpha were found responsible for the high sensitivity of this enzyme to the oxidizing agent, phenylarsine oxide. Mutation of one of these Cys residues reduced the phenylarsine oxide sensitivity of the enzyme to the same level observed with the PI4KIIIbeta protein. In search of inhibitors that would discriminate between the closely related PI4KIIIalpha and -IIIbeta enzymes, the PI3Kgamma inhibitor, PIK93, was found to inhibit PI4KIIIbeta with significantly greater potency than PI4KIIIalpha. These studies should aid development of subtype-specific inhibitors of type-III PI4Ks and help to better understand the significance of localized PtdIns4P production by the various PI4Ks isoforms in specific cellular compartments.     

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.     

Coordination of Golgi functions by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.     

A unique phosphatidylinositol 4-phosphate 5-kinase is activated by ADP-ribosylation factor in Plasmodium falciparum

In eukaryotes, calcium signalling has been linked to hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The final enzyme in the synthesis of this phosphoinositide, a Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), is activated by the small G protein ADP-ribosylation factor 1 (ARF1). In mammals, the ARF-PIP5K pathway is a key regulator of cell motility, secretion and cell signalling. We report the characterisation of a unique, putative bifunctional PIP5K in the human malaria parasite Plasmodium falciparum. The protein comprises a C-terminal, functional PIP5K domain with catalytic specificity for phosphatidylinositol 4-phosphate. The recombinant enzyme is activated by ARF1 but not phosphatidic acid. The protein also incorporates an unusual N-terminal domain with potential helix-loop-helix EF-hand-like motifs that is a member of the neuronal calcium sensor family (NCS). Intriguingly, NCS-1 has been shown to stimulate phosphatidylinositol 4-phosphate synthesis by activating mammalian and yeast phosphatidylinositol 4-kinase β in vitro in a calcium-dependent manner. The unexpected physical attachment of an NCS-like domain to the plasmodial PIP5K might reflect a unique functional link between the calcium and PtdIns(4,5)P₂ pathways allowing modulation of PtdIns(4,5)P₂ production in response to changes in intracellular calcium concentrations within the parasite.     

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).     

Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization.

PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger. The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P. Recent studies with several FYVE domain proteins challenge this general concept. Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein. We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain. PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger. Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P. An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction. This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes. Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P.     

Dual control of membrane targeting by PtdIns(4)P and ARF

In mammalian cells, three types of phosphatidylinositol 4-kinase (PI4K) are associated with the Golgi complex, where their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is concentrated. The role of PtdIns(4)P in this compartment and how the PtdIns(4)P-positive membrane domain is formed and maintained despite continuous membrane flow are, however, poorly understood. Recent work has shown that PtdIns(4)P and the small GTPase ARF1 function cooperatively in the recruitment of four-phosphate adaptor proteins (FAPPs) to the trans-Golgi network (TGN) and has implicated FAPPs in formation of the membrane domain and in post-Golgi trafficking.     

Purification and reconstitution of phosphatidylinositol 4-kinase from human erythrocytes.

A membrane-bound phosphatidylinositol 4-kinase (PtdIns kinase) has been purified to apparent homogeneity from human erythrocytes. Enzyme activity was solubilized from urea-KCl-stripped, inside-out membrane vesicles by 3% Triton X-100. Purification to apparent homogeneity was accomplished by cation-exchange chromatography on phosphocellulose, followed by heparin-acrylamide chromatography. This resulted in a nearly 3900-fold purification of PtdIns kinase activity to a specific activity of 44 nmol min-1 mg-1. The purified enzyme has an Mr of 59,000 on silver-stained SDS-PAGE; however, many preparations also contain 54 kDa and 50 kDa proteins which are related to the 59 kDa protein and have PtdIns kinase activity. Kinetic analysis of the PtdIns kinase indicate apparent Km values of 40 and 35 microM for phosphatidylinositol and ATP, respectively. The purified enzyme has been reconstituted into phospholipid liposomes and shown to phosphorylate phosphatidylinositol.     

Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.