The overarching goal of this Supplement request for CA74305 is to support a promising Ph.D. candidate from an under-represented group in her research training in the study of Akt regulation by protein post-translational modifications (PTMs). Akt is a protein Ser/Thr kinase and an important regulator of cell signaling pathways that control cell proliferation, growth, and metabolism with particular relevance to cancer. Understanding how Akt activation is influenced by the presence of certain PTMs will give insight into how different forms of Akt may be dysregulated in cancer. We will build on the proposed Aim 3 of the parent Grant by bringing to bear new methods to characterize the conformational impact of phosphorylated residues on the C-terminal tail of Akt and how they drive Akt activation. The use of cutting edge biochemical techniques including fluorescence anisotropy and alkaline phosphatase sensitivity can provide new insights into how phosphatidyl 3,4,5-triphosphate (PIP3) influences Akt conformation and how the N-terminus and linker region of Akt bind the tail. Another possible key player in Akt activation is ubiquitin that can be present at Lys8 or Lys14. To study the details of ubiquitin- mediated Akt activation from the biochemical perspective, we plan to develop a method to generate Akt constructs containing a ubiquitin mimic. This will assist us in obtaining a well-purified and specifically modified enzyme for kinetic and biophysical characterization. Investigating the effects of Lys ubiquitination on Akt will help clarify how Akt stability, activation, and cellular localization may be impacted by this PTM. We will also use protein microarrays to investigate protein substrate specificity driven by various Akt PTMs. This investigation will reveal important information that when validated with cell based studies, will be an important step forward towards finding novel vulnerabilities for cancer therapeutics. The training plan outlined here will allow the candidate to develop the experimental and professional skills to become a future chemical biology leader in the field of cancer.

Public Health Relevance

This research project will investigate the regulation of Akt, an important enzyme in the cell signaling pathway. By understanding the different mechanisms of Akt activation and the downstream proteins that are affected by each of these, we can contribute to discovering novel therapeutic agents against certain types of cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA074305-24S1
Application #
9675804
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Ogunbiyi, Peter
Project Start
1997-04-01
Project End
2023-05-31
Budget Start
2018-08-01
Budget End
2019-05-31
Support Year
24
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
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
Kalin, Jay H; Wu, Muzhou; Gomez, Andrea V et al. (2018) Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors. Nat Commun 9:53
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
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
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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