Publications

• Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIalpha at the trans-Golgi network.

  Minogue S., Chu K.M., Westover E.J., Covey D.F., Hsuan J.J., Waugh M.G.

  Type II phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIalpha has been shown to have important roles in neuronal survival and tumo ... >> More

  Type II phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIalpha has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIalpha mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIalpha activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIalpha to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIalpha demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIalpha was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIalpha. Further measurements revealed that the reduction in the mobile fraction of PI4KIIalpha correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion. << Less

  J. Lipid Res. 51:2314-2324(2010) [PubMed] [EuropePMC]

• The crystal structure of the phosphatidylinositol 4-kinase IIalpha.

  Baumlova A., Chalupska D., Rozycki B., Jovic M., Wisniewski E., Klima M., Dubankova A., Kloer D.P., Nencka R., Balla T., Boura E.

  Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2 ... >> More

  Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane. << Less

  EMBO Rep. 15:1085-1092(2014) [PubMed] [EuropePMC]

• Molecular insights into the membrane-associated phosphatidylinositol 4-kinase IIalpha.

  Zhou Q., Li J., Yu H., Zhai Y., Gao Z., Liu Y., Pang X., Zhang L., Schulten K., Sun F., Chen C.

  Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structur ... >> More

  Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the 'integral' membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα's activity. We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment. << Less

  Nat. Commun. 5:3552-3552(2014) [PubMed] [EuropePMC]

• Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.

  Nickels J.T. Jr., Buxeda R.J., Carman G.M.

  A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromat ... >> More

  A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo. << Less

  J Biol Chem 267:16297-16304(1992) [PubMed] [EuropePMC]

• Cloning and characterization of a human phosphatidylinositol 4-kinase.

  Wong K., Cantley L.C.

  Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first committed step in the biosynthesis of phosphatidylinositol 4,5-bisphosphate. Here we report the first mammalian cDNA clone of a PtdIns 4-kinase (named PI4K alpha). The 2.6-kb cDNA encodes a protein of 854 amino acids that is highly homolog ... >> More

  Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first committed step in the biosynthesis of phosphatidylinositol 4,5-bisphosphate. Here we report the first mammalian cDNA clone of a PtdIns 4-kinase (named PI4K alpha). The 2.6-kb cDNA encodes a protein of 854 amino acids that is highly homologous to the recently cloned yeast PtdIns 4-kinase STT4 and is also homologous to a second yeast PtdIns 4-kinase, PIK1. PI4K alpha has more distant sequence homology to the catalytic domains of mammalian and yeast PtdIns 3-kinases and to the yeast Tor family of proteins. It also has a region of similarity to pleckstrin homology domains and a potential ankyrin repeat. Cross-hybridizing messages were detected in all human tissues investigated. The enzymatic properties of the protein expressed in insect cells are characteristic of type II PtdIns 4-kinases (activated by detergent and inhibited by adenosine), and PI4K alpha is recognized by an antibody specific for type II PtdIns 4-kinases. << Less

  J. Biol. Chem. 269:28878-28884(1994) [PubMed] [EuropePMC]

• Cloning of a human type II phosphatidylinositol 4-kinase reveals a novel lipid kinase family.

  Minogue S., Anderson J.S., Waugh M.G., dos Santos M., Corless S., Cramer R., Hsuan J.J.

  Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so ... >> More

  Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIalpha and a second human isoform, type IIbeta. The type IIalpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes. << Less

  J. Biol. Chem. 276:16635-16640(2001) [PubMed] [EuropePMC]

• Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

  Suer S., Sickmann A., Meyer H.E., Herberg F.W., Heilmeyer L.M.G. Jr.

  Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic bead ... >> More

  Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain. << Less

  Eur. J. Biochem. 268:2099-2106(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein.

  Klima M., Toth D.J., Hexnerova R., Baumlova A., Chalupska D., Tykvart J., Rezabkova L., Sengupta N., Man P., Dubankova A., Humpolickova J., Nencka R., Veverka V., Balla T., Boura E.

  Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. ... >> More

  Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. << Less

  Sci. Rep. 6:23641-23641(2016) [PubMed] [EuropePMC]

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

  Gehrmann T., Guelkan H., Suer S., Herberg F.W., Balla A., Vereb G. Jr., Mayr G.W., Heilmeyer L.M.G. Jr.

  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 ... >> More

  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. << Less

  Biochim. Biophys. Acta 1437:341-356(1999) [PubMed] [EuropePMC]

• A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans.

  Barylko B., Gerber S.H., Binns D.D., Grichine N., Khvotchev M., Suedhof T.C., Albanesi J.P.

  Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis. Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, ap ... >> More

  Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis. Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes. However, only one of these two enzymes has been molecularly characterized. We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism. These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family. The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide. Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation. Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule. << Less

  J. Biol. Chem. 276:7705-7708(2001) [PubMed] [EuropePMC]