Chemical inducers of dimerization (CID) represent an important tool that has been implemented in numerous biological applications namely protein functions, protein stability, signal transduction, gene transcription, etc. Most generally CIDs are defined as bivalent molecules capable of inducing proximity between two targeted proteins. This proximity can in turn promote or disfavor a certain biological activity. Cell permeable small molecules in particular represent a very effective method to induce precise temporal and spatial control over a specific biological target. Our lab has devoted much effort in studying and elucidating the activity and functions of protein kinases, which represent a very attractive therapeutic target for the treatment of cancer and many other disorders. Towards this goal we have developed a general CID enabled three-hybrid split-luciferase methodology for the investigation of kinase-inhibitor interactions in vitro. We demonstrate that by modulating the kinase-ligand affinity of the CID we are able to successfully profile many structurally non-related protein kinases. We also investigate the use of weaker affinity kinase ligands to allow competitive displacement of CID by the selected inhibitor. In addition we report the design, synthesis and applications of novel CID's for the profiling of kinase inhibitors in mammalian cells and we demonstrate the feasibility of the assay to be used as a new platform for the discovery of cell permeable kinase inhibitors. Finally, we report a new ligand-gated split-kinase that can be selectively activated by photocleavable inducers of dimerization. We further prove how the activity of split-proteins can be deactivated with temporal control with use of non DNA damaging UV radiation.

The function of most proteins is regulated by post-translational modifications, of which phosphorylation in particular has been shown to be ubiquitous and of paramount importance to cell signaling. Two enzyme families, protein kinases and phosphatases, regulate phosphorylation, and aberrant activities of family members have been implicated in many diseases such as cancer and neurological disorders. Thus, understanding the function of these enzymes in living cells is important for understanding their biology and for designing new therapies, but a challenging task due to their highly conserved architecture. The major focus of the dissertation is on the development of a new approach to selectively turn-on multiple specific kinases and/or phosphatases using orthogonal ligands as chemical inducers of dimerization (CIDs). Specific kinases or phosphatases were dissected at particular sites into two inactive fragments or split-proteins. The split fragments are attached to interacting protein pairs of CID systems, such that upon addition of the specific ligand they heterodimerize with subsequent reassembly of the split-protein and concomitant activity. We demonstrated the in vitro and in cellulo feasibility of this approach using three orthogonal CIDs, rapamycin, abscisic acid, and gibberellic acid, to turn-on members of the tyrosine kinase group such as Lyn and Src, and of the tyrosine phosphatase group such as PTP1B and SHP1. We have also developed a new synthetic photocleavable di-trimethoprim CID that allows for ligand-gated turn-on of desired kinases in live cells. The new CID can be cleaved or turned-off by UV irradiation which results in a turn-off of kinase activity. Small molecule controlled split-proteins allow for developing logic gates and we demonstrate that the systems we have developed can be used to construct 7 out of the 10 basic, circuit-type Boolean phosphorylation-based logic gates in living cells. These post-translational logic gates may have interesting applications in synthetic biology. Finally, we present an initial approach to use redesigned kinases and redesigned ligands as potential scaffolds for developing new CIDs. Thus, we provide and extend new methodologies that potentially allow for posttranslational control over the activity of user defined split-kinases and split-phosphatases for interrogating and redesigning signaling pathways. The last section of this work focuses on understanding small-molecule selectivity toward protein kinases. We systematically analyzed different reported kinase screens to further understand the reliability of large scale data in the kinome field as the design of selective inhibitors is one the most useful approaches for understanding the function of enzymes or the development of drugs in a natural setting such as a primary cell or an organism.