Ligand gated split Lysine Acetyl Transferases(KATs) and Kinases
Authors: de Silva Chandi sagarika , Castillo‐Montoya Javier , Restituyo Elizabeth , Ghosh Indraneel 
Published: June 2021

Post translational modifications such as phosphorylation, methylation, acetylation, ubiquitination control activity of many proteins. Knowledge of one third of cellular proteins are covalently modified via phosphorylation, marks it as an important post translational modification. The >500 protein kinases in humans catalyze the phosphorylation of Tyr, Ser and Thr residues on proteins and thereby control their function. The structural similarity of kinases makes it challenging to selectively turn‐on or turn‐off desired kinases using small molecule inhibitors or activators. Genetic approaches are powerful, but cells and organisms respond and adapt to long term changes in gene expression levels, thus making it challenging to understand the true role of any enzyme in time and space. Towards a potential solution to this problem, we have developed a method to control the activity of individual enzymes utilizing small molecules. This approach was successfully applied to protein kinases and phosphatases, leading to the control of their activity in vitro and in cellulo. The strategy entails sequence alignment and the identification of regions that harbor significant dissimilarities. The sites are subsequently targeted for 25 amino acid loop insertion to investigate whether a specific protein can tolerate the new loop without greatly compromising its activity. The loop insertion mutants that retain the activity are used as fragmentation sites to generate ligand‐gated split proteins. The well‐studied chemical inducer of dimerization (CID), rapamycin dependent heterodimerization of FKBP/FRB was used as a first test of a successful ligand gated split‐enzyme. We have successfully demonstrated the feasibility of this approach with members of the tyrosine kinase group. Moreover, orthogonal CIDs rapamycin and abscisic acid were successfully used for regulating the activity of Kinases. Then the activity of these two‐different split‐enzyme systems were tested in HEK293T cells. split‐protein counterparts expressing stable cell lines are used to reproduce orthogonal kinase activation accurately. Furthermore, quantitative mass‐spectrometry based phosphoproteomics experiments will be used for validating the system and will be useful for demonstrating the potential of the split‐Kinases for answering relevant biological question or creating a novel signaling system. Extending the trend, we have used this approach for Lysine Acetyl Transferases(KATs). (KATs) were formerly known as Histone Acetyl Transferases (HATs) as they acetylated histones and thereby control transcription. However, recent studies strongly suggest that the acetylation mark is far more common protein modification with possibly over 7,000 acetylated proteins in the human proteome. There are numerous KATs and currently available small molecule based inhibition methods are not uniquely specific while RNAi based gene knock down studies can fail to provide details related to the true role of any enzyme as compensatory acetylation may occur. To address this problem, we have successfully created the first generation of ligand inducible split‐KATs, GCN5 and PCAF. We are currently using this approach for designing several new KATs, Myst2 and HAT1, which belong to different families, with the intention of exploring the possibility to establish temporal and spatial control over multiple KATs simultaneously in relevant cell lines.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.