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.
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.
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