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

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P 4-kinase assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100μM. A lack of inhibition of PI5P4Kγ 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 mass spectrometry revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments, which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and β homologue to render PI5P4Kγ resistant to NIH-12848 inhibition. 10μM NIH-12848 was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells, which we show express PI5P4Kγ that increases when the cells grow to confluence and polarise. 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 ...
Source: BJ Signal - Category: Biochemistry Authors: Tags: BJ Signal Source Type: research