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

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.