Small-molecule inhibitors of protein kinases have revolutionized biomedical science. In the clinic, protein kinase inhibitors have ushered in the era of personalized medicine by transforming the standard of care for a variety of cancers. At the bench, the widespread use of protein kinase inhibitors as research tools has significantly advanced our knowledge of the roles of protein kinases in signaling pathways. However, the molecular mechanisms that regulate the function of individual kinases remain poorly understood. Given the proven clinical value of kinase-directed therapeutics, a detailed understanding of kinase regulatory mechanisms can significantly impact human health by identifying new ways to modulate kinase function. The long-term goal of this proposal is to determine how a general allosteric site regulates the substrate specificity and subcellular localization of the protein kinases. This proposal focuses on the protein kinase PDK1 where this allosteric site, known as the PIF pocket, is required for the phosphorylation of most PDK1 substrates. The PIF pocket may also promote cancer cell motility. Our lab recently identified small molecules that bind to the PIF pocket in cells. We hypothesize that these PIF-pocket ligands will disrupt the interaction between PDK1 and a subset of its substrates and effector proteins, thereby granting a rapid and reversible way to perturb PIF pocket function. The work proposed here aims to use our PIF-pocket ligands 1) to study how the PIF pocket determines the substrate specificity of PDK1 and 2) to study how the PIF pocket promotes cancer cell motility. To accomplish the first aim, we will identify PDK1 substrates by mass spectrometry and determine the sensitivity of their phosphorylation to our PIF-pocket ligands. To accomplish the second aim, we will characterize the role of the PIF pocket in the motility of highly aggressive melanoma cells grown in 3D culture. These studies will highlight the importance of this general allosteric site in regulating protein kinase function. This concept may alter the landscape of kinase drug discovery and lead to the development of a new class of kinase-directed therapies.
Protein kinases are messenger proteins that relay information throughout the cell, but during cancer these messengers are overactive. Many exciting advances in cancer therapy are based on shutting down protein kinases. We are studying a new way to correct protein kinase signaling that could lead to a new class of drugs with reduced side effects.
|Lodge, Jean M; Rettenmaier, T Justin; Wells, James A et al. (2014) FP Tethering: a screening technique to rapidly identify compounds that disrupt protein-protein interactions. Medchemcomm 5:370-375|