Conformational Control of Protein Kinases Lay abstract. Protein kinases (PKs) play an important role in healthy and diseased cell signaling. However, we do not fully understand the complex signaling of PKs because we lack tools to understand the full scope of kinase signaling. In this proposal, we develop a series of tools to study noncatalytic signaling of PKs.
Conformational Control of Protein Kinases Abstract. Protein kinases (PKs) play an essential role in cell signaling and in the regulation of key biological processes, including: proliferation, differentiation, and apoptosis. Protein kinase mediated signaling is vital for healthy (and disease) cell signaling, however, our efforts to understand these complex signaling pathways has been hampered by an underappreciation and lack of tools to study the noncatalytic functions of protein kinases. For many kinases, there is a stunning disconnect between cellular signaling when perturbed by genomic knockdown (e.g., siRNA) vs pharmacological inhibition. We believe that this disconnect between genomic and pharmacological intervention results from noncatalytic kinase functions being perturbed by genomic knockdown, but not by traditional pharmacological intervention. One salient example is c-Src, a ubiquitously expressed and membrane-associated nonreceptor kinase. c-Src has been validated as a key target for many solid tumors via genomic knockdown, however, pharmacological inhibition (both in the clinic and in pre-clinical models) leads to a divergent signaling phenotype and does not support c-Src as a therapeutic target. Toward understanding the noncatalytic roles of c-Src, we recently reported that c-Src can activate EGFR via a protein?protein interaction and that this activation has a dramatic impact on cell signaling. Many PKs, including c-Src, are multi-domain proteins with regulatory domains that function in tandem with, as well as independent to, the kinase catalytic domain. The noncatalytic functions of PKs are often dependent on binding interactions between the regulatory domains and an interacting partner (e.g., c-Src and EGFR). These protein?protein interactions are modulated by global conformational changes that alter the quaternary structure of the kinase. Despite the emerging role of noncatalytic kinase signaling, efforts to understand the importance of kinase conformation on signaling pathways have been hampered by a lack of tools to assess and modulate the global conformation of PKs. As a result, we recently developed a `selective proteolysis' technique to rapidly determine global kinase conformation that is adaptable to many PKs (including c-Src). Additionally, we recently reported that an emerging class of kinase inhibitors, termed `conformation-selective' kinase inhibitors, can modulate the noncatalytic functions of protein kinases via stabilization of specific kinase conformations. Here, we propose to use our selective-proteolysis technique to develop genetic and chemical tools to study the relationship of kinase conformation on noncatalytic kinase signaling.
