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

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.