Proteins are dynamic molecules that can adopt multiple conformations in order to accomplish their respective functions. While crystallographic studies and in vitro biochemical assays can give valuable insight into a protein's structure and function, studying the cellular function of a protein in living cells remains difficult. While a postdoctoral fellow in Dr. James Wells' laboratory at the University of California, San Francisco (UCSF), I have been developing enzymatic-tagging technologies to study protein interactions in living cells and lysates. Here I propose using enzymatic-tagging coupled with quantitative proteomics to study the dynamic signaling proteins BRAF kinase and KRAS GTPase. Mutations in the KRAS-BRAF signaling pathway are found in ~30% of human cancers. While we understand the biochemical mechanisms by which mutations activate KRAS, how these mutations alter the protein-protein interactions that KRAS makes (its interactome), and what effect these changes have on cellular signaling, remains unclear. Cancer-causing mutations in the KRAS effector protein, BRAF, typically render the kinase constitutively active and independent of KRAS regulation. Surprisingly, catalytically-dead BRAF mutants have also been implicated as drivers of oncogenesis. Additionally, it has been shown that small-molecule inhibitors of BRAF can paradoxically activate the downstream MAP kinase signaling pathway. This paradoxical activation is thought to occur via a ligand-induced BRAF dimer, but we do not know how drug binding alters BRAF's interactome. The goal of this proposal is to identify how mutations and ligands change the interactome of KRAS and BRAF in living cells.
In Aim 1, I will identify changes to the BRAF interactome after treatment with ATP-competitive inhibitors.
In Aim 2, I will study the impact of clinically observed mutations on the KRAS interactome. Finally, in Aim 3, I will study the interplay between kinase-dead BRAF and mutant KRAS. Completion of these aims will expand our knowledge of KRAS and BRAF signaling in oncogenesis and may lead to new strategies for the treatment of KRAS- and BRAF-driven cancers. While performing the proposed research I will continue to utilize my previous expertise while simultaneously gaining hands-on training in mass spectrometry, proteomics, systems biology, cell biology, and cancer biology. These skills will provide me with the scientific foundation to pursue my long-term research goal of understanding how specific conformational states of signaling proteins relate to their cellular function. In addition, the advice and training I will receive from my mentor Dr. Wels and the rest of my advisory committee during the mentored phase of this award will help me to prepare for a successful career as an independent investigator. The data acquired during the mentored phase of this award will serve as preliminary data for my initial round of grant applications as an independent investigator, greatly increasing my probability of success. Completion of the research and training proposed in this application will provide me with the expertise necessary to successfully reach my long-term career goal of leading an independent research lab.
Mutations in amino acid sequence are capable of changing the conformational state of a signaling protein, leading to misregulation of the protein's function, and in many cases, a disease state such as cancer. This project seeks to understand how specific conformations of signaling proteins relate to their cellular function, with a specific focus on the role of the protins BRAF and KRAS in cancer.
| Keedy, Daniel A; Hill, Zachary B; Biel, Justin T et al. (2018) An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering. Elife 7: |
| Hill, Zachary B; Martinko, Alexander J; Nguyen, Duy P et al. (2018) Human antibody-based chemically induced dimerizers for cell therapeutic applications. Nat Chem Biol 14:112-117 |
| Hill, Zachary B; Pollock, Samuel B; Zhuang, Min et al. (2016) Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase. J Am Chem Soc 138:13123-13126 |
