Enabling in vivo Signaling Analyses via Chemical Genetics and Nanoparticle-mediated Probe Delivery
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Cell signaling transduction is a critical cellular process for various diseases. Mechanistic understanding of functional proteins will improve our fundamental understanding of the versatile signaling modules and is also vital for further therapeutic development targeting cell signaling pathways. This study is to apply nanotechnology in developing a critical but never-been-accomplished approach: for the in vivo study of cellular signaling pathways. Protein kinases mediate cell signaling by pinching a γ-phosphate off ATP and sticking the phosphate onto serine, threonine, or tyrosine substrate proteins. This process is called the phosphorylation, an approach by which proteins transmit chemical signals from the external environment into the cell, ultimately down-regulating or up-regulating the activities of their substrates, thus to regulate many essential cellular processes such as cell cycles, division, migration, and apoptosis. The kinase-substrate interaction is a fundamental regulatory mechanism in cellular signaling transduction. Many diseases are related to over-activating or over-repressing kinases and substrates, thus offering great therapeutic opportunities. For instance, type II diabetic treatment include the regulation of overreacting AMPK protein kinases’ activities. Various cancer treatments include the control of oncogenic and tumor-suppressive kinases’ activities. The analog chemical-genetic strategy offers a powerful tool to study protein kinase activity under in vitro test tube conditions. However, significant limitations have persisted in conducting studies based on this approach in intact living cells, such as xxx and xxx. Herein, we have established a method for study under in vivo conditions, which is using the analog chemical-genetic strategy in intact living cells for studying AKT1 and AKT2 kinases. We intracellularly delivered bulk A*TP with a tagged γ-phosphate group using a nanoparticle delivery system, and uniquely transfer the γ-phosphate tag into the substrates of the AKT1 or AKT2 kinases using the analog chemical-genetic strategy. With this methodology, kinase-substrate interactions are entirely intracellular, and kinase substrates can be exclusively labeled in intact living cells with the thiophosphorylation of substrates, which differ from other normal substrates. It allows for the following purification and identification of those substrates from the kinase of interest, by immunoprecipitation method using thiophosphopeptides. Our study of RAS and AKTs is essential for cell signaling, and it presents as a “prove of concept” method for the study of various kinases targeting various disease treatments. With the help of our research, it opens up tremendous a novel investigative opportunity in cell signaling studies.