This longstanding competing renewal application CA74305 concerns our plans to apply chemical and biochemical approaches in cell signaling to enhance our understanding of protein post-translational modifications (PTMs) and the pathways involved. In the next cycle, we plan to investigate the regulation of three key enzymes, PTEN, WWP2, and AKT1 that are connected to the cancer promoting phospholipid, PIP3 (phosphatidyl 3,4,5-triphosphate). PTEN is one of the most commonly mutated tumor suppressor genes and catalyzes the conversion of PIP3 to PIP2 and in this way antagonizes the action of the PI3-kinase oncogenic signaling pathway. WWP2 is a HECT domain E3 ligase (ubiquitin transferase) that targets PTEN for destruction and is considered to be an oncogene. AKT1, also an oncogene, is a protein Ser/Thr kinase that is activated by PIP3 and drives tumor formation and is an intensively studied cancer therapeutic target. PTEN, WWP2, and AKT1 are each subjected to multiple phosphorylations that are proposed to have important regulatory implications. Our research program will continue to adapt and employ protein semisynthetic methods including the newly described enzyme-catalyzed expressed protein ligation (EPL) to generate these signaling enzymes containing the requisite PTMs and appropriate mimics to interrogate how these phosphorylations influence the structure and function of these key cancer-related enzymes. Our three specific aims are: 1. Optimize enzyme-catalyzed expressed protein ligation (EPL) for protein semisynthesis and apply to the study of PTEN regulation; 2. Elucidate the molecular basis of WWP2 E3 ubiquitin ligase regulation; 3. Determine the role of C-terminal phosphorylation of AKT1 on its structure, activity, mechanism of regulation, substrate specificity, and inhibitor sensitivity. With site-specifically modified signaling enzymes in hand, we will integrate kinetic analysis, X-ray crystallography, protein microarrays, and cell-based studies to clarify key regulatory and signaling features. Upon completion of this research effort, we should have a much clearer understanding of the PIP3 signaling network. Moreover, these studies should pave the way for new therapeutic strategies to combat PTEN-AKT1 pathway dysregulation in cancer.

Public Health Relevance

These studies will investigate how three key cell signaling pathway genes, PTEN, WWP2, and AKT1, are regulated. This proposal intends to identify how abnormal regulation of these genes may contribute to cancer growth and how this cancer signaling pathway may be best targeted for therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Fu, Yali
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Brigham and Women's Hospital
United States
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Weiser, Brian P; Rodriguez, Gaddiel; Cole, Philip A et al. (2018) N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions. Nucleic Acids Res 46:7169-7178
Wang, Zhihong; Kim, Min-Sik; Martinez-Ferrando, Isabel et al. (2018) Analysis of Cellular Tyrosine Phosphorylation via Chemical Rescue of Conditionally Active Abl Kinase. Biochemistry 57:1390-1398
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Chu, Nam; Salguero, Antonieta L; Liu, Albert Z et al. (2018) Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis. Cell 174:897-907.e14
Dempsey, Daniel R; Cole, Philip A (2018) Protein Chemical Approaches to Understanding PTEN Lipid Phosphatase Regulation. Methods Enzymol 607:405-422
Dempsey, Daniel R; Jiang, Hanjie; Kalin, Jay H et al. (2018) Site-Specific Protein Labeling with N-Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms. J Am Chem Soc 140:9374-9378
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
Esadze, Alexandre; Rodriguez, Gaddiel; Weiser, Brian P et al. (2017) Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells. Nucleic Acids Res 45:12413-12424

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