Conformational Control of Protein Kinases Abstract. Kinase signaling is often oversimplified by focusing only on the downstream phosphorylation networks catalyzed by protein kinases (PKs). This traditional approach to kinase signaling ignores the noncatalytic functions that are critical for cellular signaling in both healthy and disease cells. Many PKs contain regulatory domains (e.g., SH2 and SH3 domains) that function both in tandem with, as well as independent to, the kinase catalytic domain. The noncatalytic functions of PKs are typically dependent on protein–protein interactions between the kinase’s multi-domain architecture and an interacting protein (often other kinases or scaffolding proteins). Moreover, these protein–protein interactions are regulated by large conformational changes to the quaternary structure of the protein kinase. There is evidence that noncatalytic functions of protein kinases are essential to cell signaling, however, these functions remain underappreciated due to a lack of genetic and chemical tools available to study them. 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. During the previously funded period, we developed a ‘selective proteolysis’ methodology to characterize the quaternary conformation of protein kinases. This method was quickly adopted by academics and the pharmaceutical industry; however, the method relies on the ability of thermolysin to cleave the SH2-KD linker within a kinase and not all kinases have this linker with the appropriate recognition sequence. Here, we propose a cell-based biosensor that reports on kinase conformation. Using our kinase conformation biosensors, we propose to study the role of protein conformation in single-point mutations that lead to resistance to clinical kinase inhibitors. In addition to providing information on the conformational impact of drug resistant mutations, the proposed studies will provide critical information to understand the conformational regulation of Abl, BTK, BRAF, and EGFR kinases. Finally, we propose to study the interplay of allosteric and ATP-site inhibitors and to develop multivalent kinase inhibitors for Abl and c-Src kinases. The multivalent inhibitors will be constructed from matched-conformation allosteric and ATP-site inhibitors. This proposal will develop novel bioassays to study protein kinase conformation in cellulo, to better understand drug resistance in protein kinases, and to develop novel inhibitors to study the role of conformation in kinase signaling.