Clarke Jonathan H, Giudici Maria-Luisa, Burke John E, Williams Roger L, Maloney David J, Marugan Juan, Irvine Robin F
*Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, U.K.
†MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, U.K.
Biochem J. 2015 Mar 1;466(2):359-67. doi: 10.1042/BJ20141333.
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. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100 μM. 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 (HDX)-MS (HDX-MS) 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 NIH-12848 inhibition. NIH-12848 (10 μM) 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 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 PI5P4Kγ, but not that of PI5P4Ks α or β. Overall, the data reveal a probable contribution of PI5P4Kγ 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.
NIH-12848（NCGC00012848-02）是一种假定的磷脂酰肌醇5-磷酸4-激酶γ（PI5P4Kγ）抑制剂，被用作研究这种神秘的、低活性脂质激酶的工具。体外PI5P4K分析表明，NIH-12848抑制PI5P4Kγ的IC50约为1 μM，但在浓度高达100 μM时不抑制α和β PI5P4K亚型。对PI5P4Kγ ATP酶活性缺乏抑制作用表明，NIH-12848不与该酶的ATP结合位点相互作用，使用氢-氘交换质谱（HDX-MS）直接探索结合情况，结果显示PI5P4Kγ的假定PI5P结合位点可能是相互作用区域。一系列突变实验证实了这一点，这些实验导致鉴定出一个单一的PI5P4Kγ氨基酸残基，将其突变为PI5P4Kα和β同源物可使PI5P4Kγ对NIH-12848的抑制产生抗性。将NIH-12848（10 μM）应用于培养的小鼠主肾皮质集合管（mpkCCD）细胞，我们发现这些细胞表达PI5P4Kγ，当细胞生长至汇合并极化时，PI5P4Kγ表达增加。NIH-12848抑制了mpkCCD细胞生长至汇合时发生的Na⁺/K⁺-ATP酶向质膜的转位，并且可逆地阻止了它们在培养皿上形成“穹顶”。PI5P4Kγ的特异性RNA干扰敲低模拟了NIH-12848诱导的这两种效应，但PI5P4Kα或β的敲低则没有。总体而言，数据揭示了PI5P4Kγ对上皮细胞功能极性的发展和维持可能具有的作用，并表明NIH-12848是探索PI5P4K细胞生理学的一种潜在有力工具。
