The function of phosphatidylinositol 5-phosphate 4-kinase ? (PI5P4K?) explored using a specific inhibitor that targets the PI5P-binding site.

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

SUBMITTER: Clarke JH 

PROVIDER: S-EPMC4687057 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature