title: The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site.
abstract: NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100 μM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen-deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and β homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 μM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na⁺/K⁺-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of 'domes' on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or β. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.
authors.affiliation: *Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, U.K.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Clarke
authors.affiliation: *Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, U.K.
authors.forename: Maria-Luisa
authors.initials: ML
authors.lastname: Giudici
authors.affiliation: †MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, U.K.
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.affiliation: †MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, U.K.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
authors.affiliation: ‡National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.affiliation: ‡National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
authors.forename: Juan
authors.initials: J
authors.lastname: Marugan
authors.affiliation: *Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, U.K.
authors.forename: Robin F
authors.initials: RF
authors.lastname: Irvine
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects*; D041961:Deuterium Exchange Measurement; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007672:Kidney Cortex / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D050505:Mutant Proteins; C000596980:NIH-12848; D018129:Phosphatidylinositol Phosphates; D011799:Quinazolines; D011994:Recombinant Proteins; D013876:Thiophenes; C113567:phosphatidylinositol 5-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C546220:Pip4k2c protein, mouse