Phosphorylation is a ubiquitous post-translational protein modification that plays a critical role in a variety of cellular functions. In fact, various drugs targeting the kinases that phosphorylate protein substrates have been developed to treat diseases from cancers to Alzheimer's disease. As a result, the monitoring of kinases and their phosphoprotein substrates in cells represents an important goal towards characterizing healthy and disease states and development of new drugs. Unfortunately, available methods to monitor cellular phosphorylation are challenging. New methods for monitoring and charactering kinases and their modified substrates are necessary to allow a full understanding of cellular signaling and disease formation. The long-term goal of this project is a rigorous characterization of kinase activity and kinase-catalyzed modifications. The scientific foundation of the project is our recent revelation that kinases promiscuously accept ?-phosphate modified ATP analogs as cosubstrates. Based on kinase ATP cosubstrate promiscuity, we are pursuing two significant aims. First, we hypothesize that kinases catalyze protein modifications in addition to phosphorylation. Our hypothesis is based on the fact that several ?-phosphate modified ATP analogues are naturally occurring and associated with cell stress conditions, although their mechanisms of action are unknown. Consistent with our hypothesis, we have recently discovered a novel post-translational protein modification, termed nucleotide phosphorylation. With their key role in cell signaling, these studies are critical to illuminate the overall role of kinases in cell biology. Our second significant aim centers on experiments to label kinase substrates with various functional probes to allow detection, sequence identification and characterization of the kinase-catalyzed modifications. By using the developed chemical approaches to monitor and characterize the role of kinases in healthy and diseased tissues, the significant outcome of the overall project is a greater understanding of signaling in disease formation. Because kinase enzymes are targeted in drug development, these studies will lead in the creation of more effective drugs influencing protein phosphorylation.
Kinase enzymes are involved in various diseases and are the target of multiple pharmaceutical drugs. Kinases phosphorylate protein substrates, making a characterization of phosphoproteins an important aspect of health-related research. The application outlines new approaches to characterizing kinase substrates and kinase-catalyzed modifications to aid in the understanding of disease and development of new treatments.
| 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 |
| 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 |
| 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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