Application of a Chemical Biology Toolkit to Decipher Kinase Activity 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 involves 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 regulatory protein phosphorylation events in the cells of all animals and plants. The widespread role of kinases in cell regulation is illustrated by the existence of many distinct kinases 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. Notably, kinases often carry out exactly the same function in different organisms. Despite their obvious importance, our understanding of the precise functions of kinases in living cells remains very limited. In fact, to date much of our 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 application of two complementary strategies to define the precise actions of specific kinases in living cells. We will continue our studies with a kinase known as CK2 where our lab has established a leadership position that enables us to perform studies not possible elsewhere. Our first strategy will exploit sensors that we have designed to undergo changes in fluorescence when they are phosphorylated by CK2. By introducing these sensors into living cells, we will use time-lapse fluorescence microscopy to monitor the spatiotemporal dynamics of CK2 activity during different stages in the life of cells (ie. we will determine when and where CK2 is active within living cells). To complement these studies, we have also engineered mutants of CK2 that can be selectively targeted by precisely-designed chemicals so that these chemicals can be used to investigate the precise role(s) of CK2 in specific cellular processes. Collectively, our studies will enable us to identify using living cells cellular events that are directly regulated by CK2. Given the similarity of CK2 to other members of the kinase family, our approaches can be readily translated to investigation of other kinases. Since it is evident that protein phosphorylation is a universal regulatory mechanism in all living cells, this information is critical to our understanding of how all plants and animals respond to changes in the environment or in their own fitness.