The goal of the funded project was to establish novel assays to probe the activity and substrates of polynucleotide and lipid kinases. Kinases are biologically important enzymes that catalyze the transfer of a phosphate group to substrates, which is a key chemical trigger in metabolism, cell signaling, communication, and adaptation. Importantly, kinases mediate their influence on biological event through a wide variety of substrates in vivo, including proteins, nucleic acids, lipids, and metabolites. Historically, studies involving protein kinases have played a prominent role in dissecting the biochemistry and cell biology of many biological processes. However, the paucity of chemical tools to study nucleotide and lipid kinases has hampered the full characterization of these enzymes in biological events. The goal of this funded project was to develop new methods to probe and understand the activity of nucleotide and lipid kinases, which will reveal new biochemistry and cell biology.

The funded studies were based on the Pflum lab’s early observation that protein kinases promiscuously accept gamma-phosphate modified ATP analogs as cosubstrates, which was used to develop kinase-catalyzed labeling reactions that attach functional tags to protein substrates for subsequence analysis. To enable similar studies with nucleotide and lipid substrates, kinase-catalyzed labeling was established in this project with nucleotide and lipids kinases.  One research paper was published documenting kinase-catalyzed labeling with polynucleotide kinases. A second paper is in preparation that will report the labeling with sphingosine lipid kinase and its use to develop an activity assay. The significant outcome of this funded project is the first evidence that new methods can be developed using ATP analogs and kinase-catalyzed labeling reactions. These novel chemical tools will encourage a deeper study of lipids and nucleotides in biological systems, which will enhance biochemistry and cell biology research.

Beyond the impact to the biochemistry and cell biology communities, this program also encouraged the next generation of scientists by exposing students of all ages to research. Undergraduate and graduate students performed all experiments in the project. At the middle and high school levels, the PI introduced students from the diverse Detroit metropolitan area, particularly women and underrepresented minorities, to multidisciplinary science and scientific careers through outreach programs. By integrating research and educational activities, this application has broadly impacted the community by encouraging the next generation of scientists.

Last Modified: 08/30/2018
Modified by: Mary Kay H Pflum