Protein kinase signaling is fundamental to regulating cellular homeostasis in the in every aspect of life. Protein kinases act by placing a phosphate group on a very specific subset of cellular proteins. The addition of a negative charged group on the surface of a protein can dramatically affect the folding, subcellular localization and activity of substrate proteins. Understanding the kinase responsible for a specific phosphorylation event has proven difficult as there are more than 500 mammalian kinases and greater than 50,000 phospho sites. The Shokat lab has pioneered a technique in which a particular kinase is engineered to accept either a specific inhibitor or modified substrate, which is normally not used by any other kinase. In this thesis we demonstrate the utility of this technique, by demonstrating its application to two important kinases. One of these kinases, NDR, is shown to be important in normal brain development, and we identify novel substrates that act directly downstream of NDR. Additionally, we identify novel targets of the kinase Aurora B, a kinase that is critical to normal chromosome segregation during cell division.
In addition to these well-behaved kinases, we investigate a kinase, PINK1,that proves an exception to normal kinases. We show that PINK1 has the ability to utilize a kinetin triphosphate (KTP) catalytically better than its endogenous substrate, which we therefore call a neo-substrate. The development of a neo€-substrate or new substrate for the kinase PINK1 enhances the cellular activity of the disease-associated allele of PINK1 (G309D), effectively reversing the deleterious effect of the mutation. Since PINK1 is a key element in protection against mitochondrial damage and is genetically linked to Parkinson's Disease, our approach also has immediate therapeutic implications. Typically, overactive kinases such as oncogenic kinases provide the only opportunity for drug development because inhibitors of kinases are the only modality available for regulation of kinases. Therefore we believe this may represent a novel modality to regulate the activity of kinases.