Phosphatidylinositol 4-kinase Isoform Research Articles

Phosphatidylinositol 4-kinase β is required for the ciliogenesis of zebrafish otic vesicle

The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.

Binding of Elongation Factor eEF1A2 to Phosphatidylinositol 4-Kinase β 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.

Recoverin Binds Exclusively to an Amphipathic Peptide at the N Terminus of Rhodopsin Kinase, Inhibiting Rhodopsin Phosphorylation without Affecting Catalytic Activity of the Kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.

Conservation of Regulatory Function in Calcium-binding Proteins: HUMAN FREQUENIN (NEURONAL CALCIUM SENSOR-1) ASSOCIATES PRODUCTIVELY WITH YEAST PHOSPHATIDYLINOSITOL 4-KINASE ISOFORM, Pik1

Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic alpha-helix and that frequenins bind to one side of this alpha-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.

Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain.

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kalpha1 and AtPI4Kbeta1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kalpha1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kalpha1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kalpha1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kalpha1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kalpha1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4Kbeta1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kalpha1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kbeta1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kalpha1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kalpha1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.

Mammalian phosphatidylinositol 4-kinases.

Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm.

Characterization of Type II Phosphatidylinositol 4-Kinase Isoforms Reveals Association of the Enzymes with Endosomal Vesicular Compartments

Phosphorylation of phosphatidylinositol (PI) to PI 4-phosphate is one of the key reactions in the production of phosphoinositides, lipid regulators of several cellular functions. This reaction is catalyzed by multiple enzymes that belong either to the type II or the type III family of PI 4-kinases. Type III enzymes are structurally similar to PI 3-kinases and are sensitive to PI 3-kinase inhibitors. In contrast, the recent cloning of the first type II PI 4-kinase enzyme defined a novel enzyme family. Here we characterize a new member of this family, the type IIbeta enzyme that has been identified in the NCBI data base based on its homology to the first-cloned type IIalpha enzyme. The type IIbeta enzyme has a primary transcript size of approximately 3.8 kb and shows wide tissue distribution. It contains an open reading frame of 1.4 kb, encoding a protein of approximately 54 kDa. Sequence comparison reveals a high degree of similarity to the type IIalpha enzyme within the C-terminal catalytic domain but significantly lower homology within the N-terminal region. Expression of both enzyme yields increased PI 4-kinase activity that is associated with the microsomal membrane fractions and is significantly lower for the type IIbeta than the type IIalpha form. Both enzymes use PI as their primary substrate and have no detectable activity on PI monophosphates. Epitope-tagged as well as green fluorescent protein-tagged forms of both enzymes localize primarily to intracellular membranes and show prominent co-localization with early endosomes and recycling endosomes but not with the Golgi. These compartments participate in the processing of both the transferrin receptor and the G protein-coupled AT(1A) angiotensin receptor. Our data indicate the existence of multiple forms of type II PI 4-kinase in mammalian cells and suggest that their functions are related to the endocytic pathway.

Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae.

The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.

Immunohistochemical localisation of two phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, in the central nervous system of rats.

The distribution and cellular localisation of the phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, that are believed to play important roles in the intracellular signalling mechanisms were studied in the rat brain (cortex, cerebellum, hippocampus and spinal cord) using immunocytochemistry with light and electron microscopy. PI4K230 was detected with a specific antibody purified by affinity chromatography from the egg yolk of chicken immunised with a 33-kDa fragment of bovine PI4K230, comprising amino acids 873-1175 of the native protein. PI4K92 was immunostained with a commercially available antibody raised in rabbit against amino acid residues 410-537 of human PI4K92. At the light microscopic level, the immunostaining of PI4K230 and PI4K92 showed a very similar distribution throughout the neurons and appeared as dense punctate labelling in the cytoplasm of perikarya and stem dendrites of various neurons. In addition to neurons, a strongly stained cell population was observed in the molecular layer of the cerebellar cortex that resembled Bergmann glia cells. Electron microscopy of neurons in the ventral horn of the spinal cord showed dense granular immunoprecipitates for both PI4K230 and PI4K92, mostly associated with the outer membrane of mitochondria and membranes of the rough endoplasmic reticulum. In addition, immunostaining of PI4K92 was also frequently found on the outer surface of cisterns and vesicles of Golgi complexes, whereas PI4K230 immunoreactivity was colocalised with some multivesicular bodies. Neither nuclear localisation nor a regular attachment to the cell membrane of these enzymes were observed. Our findings indicate that PI4K230 and PI4K92 are not involved directly in the ligand-stimulated turnover of phosphoinositides at the plasma membrane of neurons. However, they may provide regulatory phosphoinositides for intracellular vesicular traffic being associated with various organelles.

Evidence for phosphatidylinositol 4-kinase and actin involvement in the regulation of 125I-beta-nerve growth factor retrograde axonal transport.

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled betaNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I-betaNGF retrograde transport. PAO (20 nM to 200 microM) also inhibited NGF-dependent survival of both sympathetic and sensory neuronal populations. F-actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-betaNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-betaNGF retrograde axonal transport in vivo.