This proposal is a competing renewal application of R01 CA074305 that combines the use of chemical approaches, enzymologic measurements, cellular analysis, and proteomics to enhance our understanding of protein kinases and phosphorylation in signaling. It is now well-accepted that protein kinases through protein phosphorylation can drive a broad range of cellular growth, differentiation, and motility pathways in normal and disease processes including cancer. Although our understanding of the mechanisms and functions of kinases and their regulation has increased dramatically over the past few decades, there are still large gaps in our understanding of how they drive particular biological and pathological outputs. Filling these knowledge gaps has the potential to provide a clearer understanding of basic biomedical processes and has the opportunity to enhance the development of novel therapeutic approaches and disease diagnostic strategies. There are three Specific Aims. 1. Clarify the mechanisms of oncogenic mutation and protein interactions in EGFR (Epidermal Growth Factor Receptor) tyrosine kinase signaling. We will use our newly developed approach to generate near full-length EGFR mutant proteins to understand their enzymologic features, inhibitor sensitivities, and interactions with the tumor suppressor Mig6. 2. Elucidate the molecular mechanisms of regulation of the tumor suppressor PTEN by C-terminal phosphorylation. Using expressed protein ligation, we will build on preliminary data to generate and analyze phosphorylated forms of PTEN with respect to understanding their altered catalytic activity and conformation. 3. Illuminate the regulatory roles of acetylation of protein kinases CK2 and protein kinase A. We will employ a newly developed method to install methylthiocarbamyl (MTC) acetyl-Lys mimics by Cys alkylation and analyze the biochemical effects. Taken together, we believe these aims have the potential to enhance our understanding of how key signaling molecules are regulated by mutation or post-translational modification and identify new therapeutic opportunities for cancer and other diseases.
This proposal promises to clarify important mechanistic features of proteins that play major roles in cancer and other diseases. It employs emerging chemical technologies along with cellular and biophysical methods to fill the gaps in our knowledge about cancer signaling networks. New insights from these studies may help shape the discovery and application of the next generation of anti-cancer drugs and diagnostic strategies.
|Rodriguez, Gaddiel; Esadze, Alexandre; Weiser, Brian P et al. (2017) Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation. ACS Chem Biol 12:2260-2263|
|Boija, Ann; Mahat, Dig Bijay; Zare, Aman et al. (2017) CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II. Mol Cell 68:491-503.e5|
|Weiser, Brian P; Stivers, James T; Cole, Philip A (2017) Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis. Biophys J 113:393-401|
|Chen, Zan; Jiang, Hanjie; Xu, Wei et al. (2017) A Tunable Brake for HECT Ubiquitin Ligases. Mol Cell 66:345-357.e6|
|Mo, Gary C H; Ross, Brian; Hertel, Fabian et al. (2017) Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. Nat Methods 14:427-434|
|Henager, Samuel H; Chu, Nam; Chen, Zan et al. (2016) Enzyme-catalyzed expressed protein ligation. Nat Methods 13:925-927|
|Chen, Zan; Thomas, Stefani N; Bolduc, David M et al. (2016) Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases. Biochemistry 55:3658-66|
|Chen, Zan; Dempsey, Daniel R; Thomas, Stefani N et al. (2016) Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation. J Biol Chem 291:14160-9|
|Chu, Nam; Cole, Philip A (2015) Switching immune signals on and off. Elife 4:|
|Chen, Zan; Cole, Philip A (2015) Synthetic approaches to protein phosphorylation. Curr Opin Chem Biol 28:115-22|
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