Li Na, Zhao Youyi, Wang Danbo, Shao Shuai, Zhang Zhengyao, Liu Bo
Cancer Hospital of Dalian University of Technology, Shenyang, China.
School of Basic Medical Sciences, Faculty of Medicine, Dalian University of Technology, Dalian, China.
Biotechnol J. 2024 Dec;19(12):e202400443. doi: 10.1002/biot.202400443.
Pro-viral Insertion site for the Moloney Murine Leukemia virus 1 (PIM-1) is widely involved in various biological processes and diseases, which is based on its structure and functional sites. However, the relationship between active sites and function of PIM-1 kinase remains unclear due to the lack of effective study approaches in live cells. Herein, to visualize the effect of different active sites in PIM-1 protein on its function activity and relation with PI3K/Akt/mTOR pathway, three mutant probes of EPHY which was developed previously based on fluorescence resonance energy transfer (FRET) technology to detect PIM-1 kinase activity in living cells were further constructed and transfected into cells followed by treating with PIM-1 inhibitors, ATP and PI3K inhibitor, respectively. The results showed that Lys67 is related to substrate binding and catalytic activity of PIM-1 kinase, thereby directly regulating PI3K/Akt/mTOR signaling pathway. Pro81/Asn82 are primarily participated in PIM-1 binding to ATP, thus also involving in the modulation on PI3K/Akt/mTOR signaling pathway, but play less role in the interaction between PIM-1 protein and its substrate. Asp167 has few effects on both the catalytic function activity of PIM-1 and PI3K/AKT/mTOR pathway, even though the binding ability of PIM-1 protein to its substrate is dramatically inhibited by D167A mutation. Altogether, the mutant probes works well as visualization tools to unearth the function of active sites in PIM-1 kinase, not only facilitating the further clarification of molecular mechanism underlying PIM-1 related signaling pathways, but also shedding light on drug development and disease therapy targeting PIM-1 protein.
莫洛尼鼠白血病病毒1（PIM-1）的前病毒插入位点广泛参与各种生物学过程和疾病，这基于其结构和功能位点。然而，由于缺乏在活细胞中的有效研究方法，PIM-1激酶活性位点与功能之间的关系仍不清楚。在此，为了可视化PIM-1蛋白中不同活性位点对其功能活性的影响以及与PI3K/Akt/mTOR途径的关系，进一步构建了先前基于荧光共振能量转移（FRET）技术开发的用于检测活细胞中PIM-1激酶活性的三种EPHY突变探针，并将其转染到细胞中，然后分别用PIM-1抑制剂、ATP和PI3K抑制剂处理。结果表明，Lys67与PIM-1激酶的底物结合和催化活性相关，从而直接调节PI3K/Akt/mTOR信号通路。Pro81/Asn8主要参与PIM-1与ATP的结合，因此也参与对PI3K/Akt/mTOR信号通路的调节，但在PIM-1蛋白与其底物的相互作用中作用较小。Asp167对PIM-1的催化功能活性和PI3K/AKT/mTOR途径的影响均较小，尽管D167A突变显著抑制了PIM-1蛋白与其底物的结合能力。总之，这些突变探针作为可视化工具，能够很好地揭示PIM-1激酶活性位点的功能，不仅有助于进一步阐明PIM-1相关信号通路的分子机制，还为靶向PIM-1蛋白的药物开发和疾病治疗提供了线索。
