Design of a Chemical Biology Toolkit to Monitor Protein Kinase Function in Time and Space

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Our goal is to define how regulatory information is transmitted within cells that make up every living organism. Virtually every living process is carried out by proteins that function as enzymes to catalyze chemical reactions or transmit regulatory information, as machines or motors that generate force or enable movement, or as structural constituents that give shape and form to cells, tissues or organs, and ultimately to the entire body. Almost all cellular proteins are regulated by a process known as phosphorylation that involves chemical modification at specific sites on the protein. Consequently, this proposal will focus on cellular enzymes known as kinases that are responsible for catalyzing protein phosphorylation in the cells of all animals and plants. **The widespread role and importance of kinases in cell regulation is illustrated by two important findings. Firstly, there are many distinct kinases present in every cell. In fact, there are more than 100 different kinases present in baker's yeast and in humans, there are more than 500 different kinases. Secondly, kinases often carry out exactly the same function in different organisms. For example, several yeast kinases can be replaced with a kinase from another organism (such as a fly, mouse or human) without any undesirable consequences. Despite the obvious importance and widespread impact of protein kinases in the transmission of regulatory information, our understanding of their precise functions in living cells remains very limited. In fact, much of our current knowledge of kinases has been obtained by performing biochemical measurements in cell extracts or tissue homogenates that do not retain many of the features of living cells. **To overcome this limitation, this proposal will be focused on the development of two complementary strategies to define the precise actions of specific kinases in living cells. Initial studies will be performed with a kinase known as CK2 where our lab has established a leadership position that enables us to perform studies not possible elsewhere. Once established for CK2, we expect that our strategies can be readily adapted to investigation of other kinases. Our first strategy will involve the design of kinase mutants (designated analog-sensitive mutants) to make them susceptible to drugs (known as ATP-analogs) that do not affect any normal cellular proteins. Therefore, when we introduce the analog-sensitive kinase into cells, we will be able to use the ATP-analogs to modulate the activity of the analog-sensitive kinase without affecting any other cellular constituents. For these studies, we will also take advantage of the conserved function of kinases in yeast and in human cells. Results from initial studies in yeast will be used to guide our subsequent studies in human cells (using widely accepted human cell lines). These studies will enable us to identify - using living cells - cellular events that are directly regulated by specific kinases. The second strategy will involve the design of fluorescent sensors that can be introduced into cells to detect and monitor the activity of individual kinases within living cells. Using microscopes equipped with digital cameras, we will perform time-lapse microscopy to monitor the spatiotemporal dynamics of protein kinases in cells (ie. we will determine when and where protein kinases are active within living cells). **Collectively, these studies will provide new insights regarding the precise actions of individual protein kinases within living cells. Given the universal role of protein phosphorylation as a regulatory mechanism, this information is critical to our understanding of how all plants and animals respond to changes both within an organism and in the environment.