Related Experiment Video
Updated: Jun 4, 2025
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
Visualize PIM-1 Protein Function and Its Interaction With PI3K/Akt/mTOR Pathway Regulated by Its Active Sites Through
Na Li1,2,3, Youyi Zhao4, Danbo Wang1
1Cancer Hospital of Dalian University of Technology, Shenyang, China.
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.
Insights
Investigating active sites in PIM-1 kinase reveals Lys67 regulates substrate binding and PI3K/Akt/mTOR signaling. Pro81/Asn82 are crucial for ATP binding, while Asp167 has minimal impact on kinase activity.
Area of Science:
- Molecular biology
- Cell signaling
- Biochemistry
Background:
- The Moloney Murine Leukemia virus 1 (PIM-1) kinase is implicated in numerous biological processes and diseases.
- Understanding PIM-1's active sites is crucial for elucidating its function, but in vivo studies are limited.
Purpose of the Study:
- To visualize and delineate the roles of specific active sites within PIM-1 kinase in live cells.
- To investigate the relationship between PIM-1 active site function and the PI3K/Akt/mTOR signaling pathway.
Main Methods:
- Development and application of three mutant EPHY probes using fluorescence resonance energy transfer (FRET) technology.
- Transfection of mutant probes into cells, followed by treatment with PIM-1 inhibitors, ATP, and PI3K inhibitors.
Main Results:
- Lys67 mutation affects substrate binding and catalytic activity, directly impacting the PI3K/Akt/mTOR pathway.
- Pro81/Asn82 mutations primarily affect ATP binding, modulating the PI3K/Akt/mTOR pathway with less impact on substrate interaction.
- Asp167 mutation significantly inhibits substrate binding but has minimal effect on catalytic activity or the PI3K/Akt/mTOR pathway.
Conclusions:
- Mutant probes serve as effective tools for visualizing PIM-1 active site functions in living cells.
- Findings clarify PIM-1's molecular mechanisms and offer insights for PIM-1 targeted drug development and disease therapy.
Related Concept Videos
01:22PI3K/mTOR/AKT Signaling Pathway
01:19Protein Dynamics in Living Cells
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...