The long-term goal of this project is to develop a rapid and quantitative method for globally profiling prognostic signaling cascades in tumor cells. Protein kinases are known to participate in larger macromolecular complexes that transmit extra-cellular signals into phenotypic responses. Because these complexes act as integrators and effectors of cellular stimuli, the abundance and activity of kinases are often used as reporters for the cellular state. By selectively enriching kinase-containing signaling complexes, we will be able to dissect the molecular wiring of cancer cells. The objective of the research proposed in this application is to develop and apply a suite of affinity reagents that can be used to selectively enrich activated signaling complexes. In our first aim, we will generate bivalent affinity reagents targeting signaling complexes containing a kinase and phosphotyrosine residues. Our bivalent affinity reagent will comprise a Grb2-SH2 domain and a kinase inhibitor, which should specifically bind to phosphotyrosine residues and kinase ATP-binding sites, respectively. We will evaluate the abilities of our bivalent inhibitors to selectively enrich signaing complexes over its monovalent components using SILAC-based quantitative proteomics. These studies will guide our understanding of the modularity of our bivalent inhibitor approach and provide design principles for development of future probes. For our second aim, our goal is to design SH2-kinase inhibitors targeting distinct cell signaling pathways and evaluate their ability to enrich pathway-specific protein complexes. Our goal is to develop bivalent affinity reagents that have distinct and targeted affinities to assembled protein complexes instead of single proteins. Our application is to use kinase inhibitor-based affinity reagents to enrich kinases from biological samples overcomes the dynamic range limitations of conventional biochemical analyses. Our inhibitor- resins will be designed to enrich specific kinases in families or signalin pathways and used in combination with quantitative MS to assay both kinase abundance and activity.

Public Health Relevance

This project seeks to develop a new method to quantitatively profile signaling complexes in cancer cells and samples. Kinase signaling complexes play essential roles in cancer cell growth and proliferation. The identification of cancer-specific signaling complexes will aid in our understanding of this disease and potentially provide new cancer-specific drug targets. The technology described in this application has the potential to transform the molecular analysis of signaling pathways specific to cancer.

Agency
National Institute of Health (NIH)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA177402-02
Application #
8722501
Study Section
Special Emphasis Panel (ZCA1)
Program Officer
Knowlton, John R
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Golkowski, Martin; Brigham, Jennifer L; Perera, Gayani K et al. (2014) Rapid profiling of protein kinase inhibitors by quantitative proteomics. Medchemcomm 5:363-369
Ghosh, Rajarshi; Wang, Likun; Wang, Eric S et al. (2014) Allosteric inhibition of the IRE1? RNase preserves cell viability and function during endoplasmic reticulum stress. Cell 158:534-48