Molecular modeling and site directed mutagenesis were used to analyze the structural featuresdetermining 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 ATPyielded a PI4KIII
enzyme that showed greatly reduced wortmannin sensitivity and was catalytically stillactive. Similar substitutions were not tolerated in the type-III
enzyme rendering it catalytically inactive.Two conserved Cys residues located in the active site of PI4KIII
were found responsible for the highsensitivity of this enzyme to the oxidizing agent, phenylarsine oxide. Mutation of one of these Cys residuesreduced the phenylarsine oxide sensitivity of the enzyme to the same level observed with the PI4KIII
protein. In search of inhibitors that would discriminate between the closely related PI4KIII
and -III
enzymes, the PI3K
inhibitor, PIK93, was found to inhibit PI4KIII
with significantly greater potencythan PI4KIII
. These studies should aid development of subtype-specific inhibitors of type-III PI4Ks andhelp to better understand the significance of localized PtdIns4
P production by the various PI4Ks isoformsin specific cellular compartments.