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 PI4KIIIβ enzyme that showed greatly reduced wortmannin sensitivity and was catalytically still active. Similar substitutions were not tolerated in the type-IIIα enzyme rendering it catalytically inactive. Two conserved Cys residues located in the active site of PI4KIIIα 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 PI4KIIIβ protein. In search of inhibitors that would discriminate between the closely related PI4KIIIα and -IIIβ enzymes, the PI3Kγ inhibitor, PIK93, was found to inhibit PI4KIIIβ with significantly greater potency than PI4KIIIα. 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.
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