Live Cell Fluorogenic AKT Analysis
Introduction
Protein kinases are key cellular signaling proteins and therapeutic targets. Kinase study currently suffers insifficient live-cell research and development methods. For kinases that can be studied in live cells, the methods usually can not distinguish closely homologous protein kinases, such as Akt1 and Akt2.
Methods
We tried to develop a live cell version of the chemical genetic method developed by Dr. Kevan Shokat. The approach was to deliver the bulky ATP analog (A*TP) into cells via nano-particles. We also created fluogenic version of the A*TP analog, so that kinase-ATP binding can be monitored with fluorescent microscopes and flow cytometry analyses. Akt kinases were used as initial prototype.
Results
We were able to detect Akt substrate thio-phosphorylation in live cells. More excitingly, we were also able to detect and quantify Akt-ATP binding via activation of the fluorescence of the nano-particle-delivered fluorogenic A*TP analog.
Conclusion
Thus, a live cell fluorogenic Akt analysis approach has been established. Unlike other current methods, this approach does not rely on usage of artificial substrates as reporters, and should thus be isoform specific. The approaches should be widely applicable to other protein kinases