Visualize PIM-1 Protein Function and Its Interaction With PI3K/Akt/mTOR Pathway Regulated by Its Active Sites Through FRET Biosensors

IF 3.2 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Biotechnology Journal Pub Date : 2024-12-18 DOI:10.1002/biot.202400443
Na Li, Youyi Zhao, Danbo Wang, Shuai Shao, Zhengyao Zhang, Bo Liu
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Abstract

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.

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通过 FRET 生物传感器观察 PIM-1 蛋白的功能及其与受其活性位点调控的 PI3K/Akt/mTOR 通路的相互作用。
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来源期刊
Biotechnology Journal
Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍: Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.
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