| Reversible protein phosphorylation cascades represent a central theme in signal transduction in cells. Protein kinases are the single family of enzymes that catalyze transfer of a phosphate group from ATP to a substrate, and thus are key regulators of these pathways. Currently, drug-discovery research is increasingly focused on kinase inhibitors as molecularly targeted drugs. However, with 518 members in the human kinome, it remains a challenge to screen small molecule kinase inhibitors for absolute specificity. The goal of our research is to develop a set of molecular probes that can be used to profile the functional state of protein kinases in lysates and living cells. We have developed a general set of affinity-based probes that are selective for the kinase superfamily and are able to covalently label the active site of protein kinases. The characterization and application of these probes will be described. Of those inhibitors that that are of clinical interest, many target the Src-family kinases (SFKs), a set of nine modular, non-receptor, tyrosine kinases that regulate a variety of cellular processes. In addition to a catalytic domain, the SFKs share the regulatory SH2 and SH3 domains, and the phosphorylation state of an SFK is critical in dictating its activity. Small molecule inhibitors have been developed that target both the active and inactive conformations of SFKs, but the impact of the phosphorylation state of these kinases on inhibitor binding and potency has not been well characterized. To this end, we have also developed a cell-permeable molecular probe that binds and covalently labels the active site of the SFKs. We have used this probe as a tool to explore the binding modes of different SFK phospho-isoforms against a panel of kinase inhibitors both in vitro and in cells. With this probe we demonstrate that closely-related SFKs behave fundamentally differently with small molecule inhibitors that target inactive kinase conformations. These studies provide insight into the regulation of this important family of tyrosine kinases. |
