Project Summary / Abstract Protein kinases are ubiquitous signaling enzymes that regulate nearly every aspect of cell behavior. Kinase activity is disrupted or misregulated in countless human diseases including cancer, metabolic diseases, and neurological and developmental disorders. Kinases are regulated by upstream signals, which tune kinase enzymatic activity by means of an array of binding partners and post-translational modifications. Although in vitro reconstitution can be used to link binding partners and modifications to their effects on kinase enzymatic activity, an in vitro approach is time consuming, challenging to apply to multiple partners/modifications simultaneously, and not applicable to in vivo settings. This limitation has prevented the field from understanding how kinases integrate upstream signals to establish an appropriate level of activity. The long-term goal of the proposed research is to understand the biochemical basis of signal integration by protein kinases in vivo. To enable progress towards this goal, new experimental tools are needed because existing tools are unable to resolve the molecular state of a kinase molecule (i.e., its complement of modifications and binding partners) and simultaneously measure its activity. This project focuses on developing a single-molecule enzymatic assay for protein kinase activity. By studying single molecules, the applicants will overcome the limitations of ensemble averaging, which would otherwise preclude directly linking kinase modifications and binding partners to changes in activity because of the heterogenous nature of protein complexes in vivo. A single-molecule approach will also enable direct application to cellular protein kinases, eliminating the need for in vitro reconstitution. The central objective of this work is to gain new technical knowledge that will enable application of fluorescence-based kinase activity reporters in single molecule assays. The applicants will explore three different approaches for isolating single kinase molecules, measuring their activity and simultaneously determining which binding partners and post-translational modifications are present (Aim 1). Approaches that appear promising will then be tested on cell-derived kinase molecules to determine whether application of single-molecule kinase assays in vivo is feasible (Aim 2). The work proposed in this application is significant because it will establish the feasibility of a novel approach to measuring protein kinase activity, with the potential to ultimately yield fundamental insights into cellular signal transduction. The proposed work is innovative, in the applicant’s opinion, because it represents a fundamentally new paradigm for studying signaling by protein kinases. By establishing new tools with the potential to answer fundamental questions about signal integration by cellular protein kinases, this work will contribute to the advancement of basic biomedical research.