Kinase mediated phosphorylation of proteins broadly regulates cellular responses in normal and diseasestates. Kinases in cellular signaling networks play the role of `micro-processors' that couple different stimuli todistinct signaling outputs. The versatility and specificity of their cellular function arise from the coordination ofseveral intra-molecular and inter-molecular protein interactions. However, current approaches to probe kinasestreat 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 proteininteractions 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/therapeuticsthat differentiate between closely related kinases. We have developed and tested kinase toolboxes for focaladhesion kinase (FAK) and protein kinase C (PKC). We propose to pursue three complementary and parallelgoals in order to realize the transformative potential of this new technology, while distributing risk. Our first goalis to use the PKC toolbox to map the spatial and temporal regulation of two closely related PKC isoforms incellular models of cardiac hypertrophy and diabetic retinopathy. In addition to proof-of-concept, the PKCtoolbox 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 theregulation of five closely related AGC kinases (PKA, Akt/PKB, PKC, PDK1 and S6K1). Our third goal is toconduct pilot studies of three new approaches, based on the kinase toolbox, to design isoform-specificinhibitors 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 drugtargets in disease states such as diabetes, heart failure and cancer. Successful completion of the outlinedstudies will transform our understanding of kinases in general, while providing researchers with new tools anda roadmap to study their cellular function.

Public Health Relevance

Kinases are a family of proteins that are an integral part of cellular signaling networks. Kinases are a prominentdrug target for diseases such as diabetes; heart failure and cancer. The proposed research will use aninnovative technology to provide detailed mechanistic insights into the cellular function of kinases; along withpilot studies using transformative approaches to identify/design new therapeutics that target them.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
7DP2CA186752-02
Application #
9116553
Study Section
Special Emphasis Panel (ZRG1-MOSS-C (56))
Program Officer
Knowlton, John R
Project Start
2013-09-30
Project End
2018-08-31
Budget Start
2015-07-28
Budget End
2018-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$1,462,958
Indirect Cost
$517,112
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Sommese, Ruth F; Sivaramakrishnan, Sivaraj (2018) Engineering Synthetic Myosin Filaments Using DNA Nanotubes. Methods Mol Biol 1805:93-101
Ma, Ning; Lippert, Lisa G; Devamani, Titu et al. (2018) Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism. Biochemistry 57:6387-6390
Sommese, Ruth F; Ritt, Michael; Swanson, Carter J et al. (2017) The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKC?. J Biol Chem 292:2873-2880
Lee, Sangbae; Devamani, Titu; Song, Hyun Deok et al. (2017) Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase C?. J Biol Chem 292:16300-16309
Malik, Rabia U; Dysthe, Matthew; Ritt, Michael et al. (2017) ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells. Sci Rep 7:7749
Cho, Jung-Hwa; Swanson, Carter J; Chen, Jeannie et al. (2017) The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators. ACS Chem Biol 12:1066-1074
Gupte, Tejas M; Malik, Rabia U; Sommese, Ruth F et al. (2017) Priming GPCR signaling through the synergistic effect of two G proteins. Proc Natl Acad Sci U S A 114:3756-3761
Ritt, Michael; Sivaramakrishnan, Sivaraj (2016) Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor. Mol Pharmacol 89:407-12
Swanson, Carter J; Sommese, Ruth F; Petersen, Karl J et al. (2016) Calcium Stimulates Self-Assembly of Protein Kinase C ? In Vitro. PLoS One 11:e0162331
Semack, Ansley; Sandhu, Manbir; Malik, Rabia U et al. (2016) Structural Elements in the G?s and G?q C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling. J Biol Chem 291:17929-40

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