Kinase mediated phosphorylation of proteins broadly regulates cellular responses in normal and disease states. Kinases in cellular signaling networks play the role of 'micro-processors'that couple different stimuli to distinct signaling outputs. The versatility and specificity of their cellular function arise from the coordination of several intra-molecular and inter-molecular protein interactions. However, current approaches to probe kinases treat them as simple 'on-off'switches and do not address their complex spatial and temporal regulation in cells. We have developed a technology, termed the kinase toolbox, which monitors and/or controls these protein interactions to provide a detailed mechanistic understanding of the cellular function of any kinase. In addition, the kinase toolbox overcomes the limitations of existing techniques to identify small molecules/therapeutics that differentiate between closely related kinases. We have developed and tested kinase toolboxes for focal adhesion kinase (FAK) and protein kinase C (PKC). We propose to pursue three complementary and parallel goals in order to realize the transformative potential of this new technology, while distributing risk. Our first goal is to use the PKC toolbox to map the spatial an temporal regulation of two closely related PKC isoforms in cellular models of cardiac hypertrophy and diabetic retinopathy. In addition to proof-of-concept, the PKC toolbox has already provided us with new conceptual insights that broadly apply to the AGC kinase superfamily (60 members). Our second goal is to use these insights to understand the similarities and differences in the regulation of five closely related AGC kinases (PKA, Akt/PKB, PKC, PDK1 and S6K1). Our third goal is to conduct pilot studies of three new approaches, based on the kinase toolbox, to design isoform-specific inhibitors of AGC kinases. Taken together, the proposed research is an essential first step towards our long-term goal of designing and characterizing high-specificity inhibitors of AGC kinases, which are important drug targets in disease states such as diabetes, heart failure and cancer. Successful completion of the outlined studies will transform our understanding of kinases in general, while providing researchers with new tools and a roadmap to study their cellular function.

National Institute of Health (NIH)
National Cancer Institute (NCI)
NIH Director’s New Innovator Awards (DP2)
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Special Emphasis Panel (ZRG1-MOSS-C (56))
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Knowlton, John R
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University of Michigan Ann Arbor
Anatomy/Cell Biology
Schools of Medicine
Ann Arbor
United States
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Swanson, Carter J; Sivaramakrishnan, Sivaraj (2014) Harnessing the unique structural properties of isolated ?-helices. J Biol Chem 289:25460-7
Norris, Stephen R; Soppina, Virupakshi; Dizaji, Aslan S et al. (2014) A method for multiprotein assembly in cells reveals independent action of kinesins in complex. J Cell Biol 207:393-406
Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj (2014) Myosin lever arm directs collective motion on cellular actin network. Proc Natl Acad Sci U S A 111:4091-6
Swanson, Carter J; Ritt, Michael; Wang, William et al. (2014) Conserved modular domains team up to latch-open active protein kinase C?. J Biol Chem 289:17812-29