The activity of protein kinases are naturally gated by a variety of physiochemical inputs, such as phosphorylation, metal ions, and small molecules. In order to design protein kinases that can be gated by user-defined inputs, we describe a sequence dissimilarity based approach for identifying sites in protein kinases that accommodate 25-residue loop insertion while retaining catalytic activity. We further demonstrate that the successful loop insertion mutants provide guidance for the dissection of protein kinases into two fragments that cannot spontaneously assemble and are thus inactive but can be converted into ligand-gated catalytically active split-protein kinases. We successfully demonstrate the feasibility of this approach with Lyn, Fak, Src, and PKA, which suggests potential generality.
Protein kinases phosphorylate client proteins, while protein phosphatases catalyze their dephosphorylation and thereby in concert exert reversible control over numerous signal transduction pathways. We have recently reported the design and validation of split-protein kinases that can be conditionally activated by an added small molecule chemical inducer of dimerization (CID), rapamycin. Herein, we provide the rational design and validation of three split-tyrosine phosphatases (PTPs) attached to FKBP and FRB, where catalytic activity can be modulated with rapamycin. We further demonstrate that the orthogonal CIDs, abscisic acid and gibberellic acid, can be used to impart control over the activity of split-tyrosine kinases (PTKs). Finally, we demonstrate that designed split-phosphatases and split-kinases can be activated by orthogonal CIDs in mammalian cells. In sum, we provide a methodology that allows for post-translational orthogonal small molecule control over the activity of user defined split-PTKs and split-PTPs. This methodology has the long-term potential for both interrogating and redesigning phosphorylation dependent signaling pathways.
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.
The over 500 human protein kinases are estimated to phosphorylate at least one-third of the proteome. This posttranslational modification is of paramount importance to intracellular signaling and its deregulation is linked to numerous diseases. Deciphering the specific cellular role of a protein kinase of interest remains challenging given their structural similarity and potentially overlapping activity. In order to exert control over the activity of user-defined kinases and allow for understanding and engineering of complex signal transduction pathways, we have designed ligand inducible split protein kinases. In this approach, protein kinases are dissected into two fragments that cannot spontaneously assemble and are thus inactive. The two kinase fragments are attached to chemical inducers of dimerization (CIDs) that allow for ligand induced heterodimerization and concomitant activation of kinase activity.
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