title: Design of drug-resistant alleles of type-III phosphatidylinositol 4-kinases using mutagenesis and molecular modeling.
abstract: 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.
authors.affiliation: Section on Molecular Signal Transduction, Center for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA. ballat@mail.nih.gov
authors.forename: Andras
authors.initials: A
authors.lastname: Balla
authors.forename: Galina
authors.initials: G
authors.lastname: Tuymetova
authors.forename: Balazs
authors.initials: B
authors.lastname: Toth
authors.forename: Zsofia
authors.initials: Z
authors.lastname: Szentpetery
authors.forename: Xiaohang
authors.initials: X
authors.lastname: Zhao
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Knight
authors.forename: Kevan
authors.initials: K
authors.lastname: Shokat
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Steinbach
authors.forename: Tamas
authors.initials: T
authors.lastname: Balla
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D000483:Alleles; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D019870:1-Phosphatidylinositol 4-Kinase; D000077191:Wortmannin