Protein Tyrosine Kinase Substrate Selection, Catalytic Mechanism, and Inhibition

Cellular signalling has become a major research theme in biology and medicine over the past twenty years. The complex pathways and protein components in signal transduction are emerging with increasing clarity. Over the last 15 years, the protein tyrosine kinases have been identified as key players in cellular regulation. They are involved in immune, endocrine, and nervous system physiology and pathology and thought to be important in the development of many cancers. As such they serve as drug targets for many different diseases.

Tyrosine kinases catalyze the transfer of the gamma-phosphoryl group from ATP to tyrosine hydroxyls of proteins. This family of kinases shares amino acid sequence homology with the serine/threonine kinase family. Although the number of tyrosine kinases being discovered is growing exponentially, molecular details pertaining to their substrate recognition, catalytic mechanism, and intra- and intermolecular regulation are lacking. Our interest is in addressing these issues at a chemical level, with an ultimate aim toward inhibitor design. We plan to use the Csk (C-terminal Src Kinase), as a prototype for the initial phase of our investigation. Human Csk is a non-receptor protein tyrosine kinase. One of its major functions in vivo is to specifically phosphorylate a conserved C-terminal tyrosine on the proto-oncogene Src and Src family members (including Lck, Fyn, Yes etc.) and down-regulate them by shutting off their kinase activity. In this way, Csk plays a role in cell growth and differentiation in a variety of tissues including the immune system, bone, and the nervous system.

Our lab has begun to characterize the enzymatic properties of Csk with peptide substrates. We have shown that a ternary complex mechanism is obeyed, with random substrate binding. Using site-directed mutagenesis and chemical synthesis, we have proposed a mechanism whereby the conserved "catalytic base" asp-314 is important in active site orientation rather than deprotonating tyrosine.

Having completed these initial studies, the foundation has been established for the exciting possibilities of detailed catalytic understanding, insights into structure/function relationships, and the design of potent and specific inhibitors. By studying the phosphorylation of Lck by Csk, we will define the key domains that govern substrate selection. This will entail making deletion constructs of both Csk and Lck, overproducing the proteins, and studying their behavior in pure form. We will also synthesize Csk substrate peptides tethered to Csk docking domains with flexible linkers to serve as molecular rulers. These should define the optimal solution phase distance between docking and phosphorylation sites. We will continue to carry out studies of the Csk chemical mechanism using a combination of site-directed mutagenesis, tyrosine analogs, enzyme kinetics, and structural studies. These should help establish the role of active site residues and the nature of the phosphoryl transfer transition state. Based on our previous mechanistic studies, we have designed Csk transition state analog inhibitors. We also plan to elucidate the mechanism of action of non-peptide protein tyrosine kinase inhibitors and to develop new and more potent analogs. These studies should result in tools to dissect the roles of Src-related pathways in cells and lead compounds for novel cancer chemotherapies.

Another general puzzle in the protein kinase field is defining which particular substrates are subject to phosphorylation by specific kinases in the cell. Given the thousands of kinases and the tens of thousands of potential protein phosphorylation target sites in cells, this problem is complex, but fundamental in the unraveling of signal transduction pathways. To address this issue of substrate selection we will undertake a chemical approach. We plan to design novel cross-linking agents which can be used to identify enzyme-substrate partners.