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Abstract
Phosphatidylinositol (PtdIns) 4-kinase catalyzes the synthesis of PtdIns-4-P, the precursor of an array of lipid second messengers generated by additional phosphorylation by PtdIns-4-P 5-kinase and PtdIns 3-kinase. PtdIns 4-kinase activity is conserved from yeast to higher eukaryotes. Multiple isoforms of mammalian PtdIns 4-kinase have been purified, and the activities have been detected in almost all subcellular locations. We previously reported the cloning and characterization of the first mammalian PtdIns 4-kinase named PI4Kalpha (Wong, K., and Cantley, L. C. (1994) J. Biol. Chem. 269, 28878-28884). Alternatively spliced forms of PI4Kalpha have also been identified from several sources including bovine brain (Gehrmann, T., Vereb, G., Schmidt, M., Klix, D., Meyer, H. E., Varsanyi, M., and Heilmeyer, L. M., Jr. (1996) Biochim. Biophys. Acta 1311, 53-63). Recently we isolated a distinct human PtdIns 4-kinase gene, named PI4Kbeta, that encodes an enzyme that is wortmannin sensitive (Meyers, R., and Cantley, L. C. (1997) J. Biol. Chem. 272, 4384-4390). Here we report the locations of these enzymes and provide evidence for other yet unidentified isoforms present in specific organelles. PI4Kalpha is mostly membrane-bound and located at the endoplasmic reticulum; whereas PI4Kbeta is in the cytosol and also present in the Golgi region. Neither of these isoforms accounts for the major type II PtdIns 4-kinase activity detected in the lysosomes and plasma membrane fraction.
Highlights
Phosphatidylinositol (PtdIns) 4-kinase catalyzes the synthesis of PtdIns-4-P, the precursor of an array of lipid second messengers generated by additional phosphorylation by PtdIns-4-P 5-kinase and PtdIns 3-kinase
We assessed the isozyme specificity of the PtdIns 4-kinase antibodies by immunoblotting the recombinant PI4K␣ and PI4K that were expressed in Sf9 insect cells and in E. coli, respectively
When the blot was reprobed with anti-PI4K antibody, it reacted with the 120-kDa GST-PI4K only in cells subjected to the induction of expression and not in the nontransformed E. coli
Summary
Materials—PtdIns, [␥-32-P] ATP, and silica gel plates were purchased from Avanti (Alabaster, AL), DuPont NEN, and E. Mouse monoclonal antibody 12CA5, reactive to the influenza virus hemagglutinin, was purchased from BabCo. Rabbit polyclonal anti-PI4K␣ antibody 3334 was raised to a peptide corresponding to amino acids 501–512, KPYPKGDERKKA, coupled to keyhole limpet hemacyanin [1]. AntiPI4K antibody was raised against a GST-fusion protein that was generated by polymerase chain reaction using oligonucleotide primers that generate amino acids 410 –538 of PI4K [3]. The GST-PI4K construct was generated by polymerase chain reaction as described in detail elsewhere [3] and was expressed in Escherichia coli by standard procedures. Preparation of Particulate and Cytosolic Fractions—Adherent parental CHO-IRS or CHO-IRS cells transfected with PI4K␣ were harvested with phosphate-buffered saline containing 1 mM EDTA and 1 mM EGTA. Fixed cells were permeabilized and nonspecific reactive sites were blocked for 30 min at room temperature in phosphate-buffered saline containing 0.1% Triton X-100, 5% normal goat and donkey serum. The organic layer was collected and analyzed by both thin layer chromatography and HPLC as described in detail elsewhere [31]
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