The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase gamma (PI5P4K gamma) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4K gamma with an IC50 of approximately 1 mu M but did not inhibit the alpha and beta PI5P4K isoforms at concentrations up to 100 mu M. A lack of inhibition of PI5P4K gamma 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 PI5P4K gamma to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4K gamma amino acid residue that can be mutated to its PI5P4Ks alpha and beta homologue to render PI5P4K gamma resistant NIH-12848 inhibition. NIH-12848 (10 mu M) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4K gamma 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 PI5P4K gamma, but not that of PI5P4Ks alpha or beta. Overall, the data reveal a probable contribution of PI5P4K gamma 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.