Phosphorylation is a ubiquitous protein post-translational modification that plays a critical role in the cell signaling pathways governing cellular functions. The dynamic activities of kinase and phosphatase enzymes ensure proper phosphorylation in healthy cells. However, when phosphorylation becomes unregulated, generally through aberrant kinase or phosphatase activities, cells become diseased. In fact, various drugs targeting kinases and phosphatases have been developed to treat diseases from cancers to Alzheimer's. As a result, the monitoring of kinases and phosphatases, and the signaling pathways that they control, represents an important goal towards characterizing disease states and developing new drugs. Unfortunately, the available methods to monitor cellular phosphorylation and map the complex signaling pathways controlled by phosphorylation are challenging. New methods are necessary to fully characterize phosphorylation, cellular signaling, and disease formation. The long-term goal of this project is to rigorously characterize kinase and phosphatase activities in cells to map signaling pathways in healthy and diseased states. The scientific foundation established in the prior funding period is our revelation that kinases promiscuously accept ?-phosphoryl modified ATP analogs as cosubstrates. Based on this cosubstrate promiscuity, we pioneered several kinase-catalyzed labeling methods.
We aim here to apply kinase-catalyzed labeling to several critical challenges in cell signaling research. First, we will develop kinase-catalyzed biotinylation to distinguish dynamic versus nondynamic phosphorylation events (Specific Aim 1). Second, kinase-catalyzed crosslinking will be established to monitor transient phosphorylation-mediated protein-protein interactions that are challenging to characterize by traditional methods, focusing on kinase-substrate pairs (Specific Aim 2). Phosphorylation-dependent crosslinking has the exciting potential to provide network-level details about signal transduction pathways, which is a critical yet underdeveloped need in cell signaling studies. A bold new direction for the project is application of kinase-catalyzed labeling to Ser/Thr phosphatase substrate identification (Specific Aim 3). Few strategies are available to characterize substrates of Ser/Thr phosphatases, making our innovative approach crucial to embolden cell signaling research. By studying both kinases and phosphatases, kinase-catalyzed labeling experiments will build a more detailed and complete picture of the complex cell signaling pathways governing disease. In total, the chemical tools established in this project will provide innovative and unprecedented approaches to monitor the role of phosphorylation in biology.
Kinase and phosphatase enzymes are involved in various diseases and are the target of multiple pharmaceutical drugs. The application outlines new chemical approaches to characterizing kinase and phosphatase substrates to aid in the understanding of disease and development of new treatments.
| Fouda, Ahmed E; Embogama, D Maheeka; Ramanayake-Mudiyanselage, Vindya et al. (2018) Chitosan-assisted permeabilization of ATP-biotin for live cell kinase-catalyzed biotinylation. Biotechniques 65:143-148 |
| Dedigama-Arachchige, Pavithra M; Acharige, Nuwan P N; Pflum, Mary Kay H (2018) Identification of PP1-Gadd34 substrates involved in the unfolded protein response using K-BIPS, a method for phosphatase substrate identification. Mol Omics 14:121-133 |
| Embogama, D Maheeka; Pflum, Mary Kay H (2017) K-BILDS: A Kinase Substrate Discovery Tool. Chembiochem 18:136-141 |
| Senevirathne, Chamara; Embogama, D Maheeka; Anthony, Thilani A et al. (2016) The generality of kinase-catalyzed biotinylation. Bioorg Med Chem 24:12-9 |
| Dedigama-Arachchige, Pavithra M; Pflum, Mary Kay H (2016) K-CLASP: A Tool to Identify Phosphosite Specific Kinases and Interacting Proteins. ACS Chem Biol 11:3251-3255 |
| Fouda, Ahmed E; Pflum, Mary Kay H (2015) A Cell-Permeable ATP Analogue for Kinase-Catalyzed Biotinylation. Angew Chem Int Ed Engl 54:9618-21 |
| Garre, Satish; Senevirathne, Chamara; Pflum, Mary Kay H (2014) A comparative study of ATP analogs for phosphorylation-dependent kinase-substrate crosslinking. Bioorg Med Chem 22:1620-5 |
| Wambua, Magdalene K; Nalawansha, Dhanusha A; Negmeldin, Ahmed T et al. (2014) Mutagenesis studies of the 14 Å internal cavity of histone deacetylase 1: insights toward the acetate-escape hypothesis and selective inhibitor design. J Med Chem 57:642-50 |
| Senevirathne, Chamara; Pflum, Mary Kay H (2013) Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: implications for phosphoproteomics. Chembiochem 14:381-7 |
| Senevirathne, Chamara; Green, Keith D; Pflum, Mary Kay H (2012) Kinase-Catalyzed Biotinylation. Curr Protoc Chem Biol 4:83-100 |
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