Wednesday, May 1, 2024
5/1/2024
This project is developing live-cell kinase analysis methods that can distinguish close isoforms. Akt1 and Akt2 are our initial kinase model. The two kinases share high sequence homology, but mysteriously display functional difference in development, diabetics and et al. We aim to develop fluorescent live-cell isoform- specific Akt activity quantification, and to determine whether different Akt1 and Akt2 substrate specificity and/or preference are an underlying mechanism. The methods are widely applicable. The project will nurture the growth of many undergraduate researchers to their next stage of their biomedical career. By combining the well-established chemical-genetic method developed by Dr. Kevan Shokat and nanoparticle-delivery of the cell-impermeable bulky analog N6-benzyl-ATP-g-S (A*TP-g-S), we achieved specific substrate tagging in live cells, that is, the ability to distinguish the kinase reaction of interest from those of the > 500 kinases encoded in the human genome – a prerequisite for live-cell “kinase assay”. Briefly, according to the Shokat method, Akt1/2 gatekeeper Met (M) was mutated to Gly (G) to enlarge the ATP binding pockets to accommodate A*TP; i.e., creation of the Akt1M-G and Akt2M-G mutants. We showed that only Akt1/2M-G could efficiently use delivered A*TP-g-S due to the bulkier adenine moiety, and thiophosphate- tag their substrates. Currently, A*TP is being tagged with the MANT or TNP fluorogenic dyes. Exclusive Akt1/2M-G binding to nanoparticle-delivered MANT-/TNP-A*TP will activate their fluorescence, which in turn quantify Akt1/2 ATP-binding, and thus enzymatic, activities in intact live cells; that is, live-cell quantification of Akt1/2 kinase activities. We are also generating the mutations via genome editing, so that Akt1/2M-G genes stay in native genomic context. Our aims to accomplish the goals are: Aim 1: Fluorogenic live-cell quantification of Akt ATP binding activities via expression vectors; Aim 2: Adapting the method to prime-edited HCT116 human cells with enlarged Akt1 or Akt2 ATP binding pockets, i.e., the Akt1M¿G cells and the Akt2M¿ G cells; and Aim 3: To identify and differentiate the Akt1 and Akt2 spectrums of substrates. Significance: We are developing a widely applicable system for live-cell kinase studies. Kinase-substrate relationship analysis via substrate tagging in live cells will be established. Our fluorogenic live-cell kinase assay will bypass usage of artificial substrates to enable, among others, distinguishing homologous kinase isoforms such as Akt1 and Akt2. The results will set a new and general paradigm for studying protein kinases. Akt1 and Akt2 have long been targeted for drug and therapeutic development. Identification of their respective spectrum of substrates will help guide further development and improvement efforts for relevant human diseases. Additionally, the project provides an excellent interdisciplinary platform for students to integrate classroom education and research activities.
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