Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III protects from dephosphorylation and stabilizes lipid kinase activity

Phosphatidylinositol-4-kinase-III (PI4KIII) is activated at the Golgi compartment by PKD-mediated phosphorylation. Subsequent mechanisms responsible for continuous PtdIns(4)P production at Golgi membranes and potential interaction partners of activated PI4KIII are unknown. Here we identify phosphoserine/-threonine binding 14-3-3 proteins as novel regulators of PI4KIII activity downstream of this phosphorylation. The PI4KIII-14-3-3 interaction, evident from GST pulldowns, co-immunoprecipitations and bimolecular fluorescence complementation, was augmented by phosphatase inhibition with okadaic acid. Binding of 14-3-3 proteins to PI4KIII involved the PKD phosphorylation site Ser294, evident from reduced 14-3-3 binding to a S294A PI4KIII mutant. Expression of dominant negative 14-3-3 proteins resulted in decreased PI4KIII Ser294 phosphorylation, whereas wildtype 14-3-3 proteins increased phospho-PI4KIII levels. This was because of protection of PI4KIII Ser294 phosphorylation from phosphatase-mediated dephosphorylation. The functional significance of the PI4KIII-14-3-3 interaction was evident from a reduction of PI4KIII activity upon dominant negative 14-3-3 protein expression. We propose that 14-3-3 proteins function as positive regulators of PI4KIII activity by protecting the lipid kinase from active site dephosphorylation, thereby ensuring a continuous supply of PtdIns(4)P at the Golgi compartment.