The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site.
The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site.
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DOI：
10.1042/bj20141333
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发表时间：
2015-03-01
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影响因子：
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通讯作者：
Irvine RF
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文献类型：
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作者：
Clarke JH;Giudici ML;Burke JE;Williams RL;Maloney DJ;Marugan J;Irvine RF
NIH-12848（NCGC 00012848 -02）是一种公认的磷脂酰肌醇5-磷酸4-激酶γ（PI 5 P4 K γ）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PI 5 P4 K测定显示，NIH-12848抑制PI 5 P4 K γ，IC 50约为1 μM，但在高达100 μM的浓度下不抑制α和β PI 5 P4 K亚型。缺乏对PI 5 P4 K γ ATP酶活性的抑制表明，NIH-12848不与该酶的ATP结合位点相互作用，并且使用氢-氘交换（HDX）-MS（HDX-MS）直接探索结合揭示了PI 5 P4 K γ的推定PI 5 P结合位点可能是相互作用的区域。这一点通过一系列突变实验得到证实，这些突变实验导致鉴定出单个PI 5 P4 K γ氨基酸残基，该氨基酸残基可突变为其PI 5 P4 K α和β同源物，从而使PI 5 P4 K γ对NIH-12848抑制具有抗性。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，当细胞生长至汇合和成熟时，表达PI 5 P4 K γ。NIH-12848抑制了当mpkCCD细胞生长至汇合时发生的Na+/K+-ATP酶向质膜的移位，并且还可逆地防止了它们在培养皿上形成“圆顶”。这两种NIH-12848诱导的作用均通过特异性RNAi敲低PI 5 P4 K γ而非PI 5 P4 Ks α或β来模拟。总体而言，数据揭示了PI 5 P4 K γ对上皮细胞功能极性的发育和维持的可能贡献，并表明NIH-12848是探索PI 5 P4 K的细胞生理学的潜在有力工具。我们已经表征了酶磷脂酰肌醇5-磷酸4-激酶γ的特异性抑制剂，包括确定抑制剂结合酶的位置，然后将该抑制剂应用于肾细胞系以阐明酶的细胞内功能。
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. We have characterised a specific inhibitor of the enzyme Phosphatidylinositol 5-phosphate 4-kinase γ, including establishing where on the enzyme the inhibitor binds, and then applied this inhibitor to a kidney cell line to elucidate the intracellular functions of the enzyme.