INVESTIGATING THE DIMERIZATION OF RASG USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET)
AuthorWEISS, ETHAN T.
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PublisherThe University of Arizona.
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AbstractIn diseases such as cancer, metastasis, the spread of cancer to secondary tumor sites, is one of the greatest challenges associated with disease progression and treatment. Ras has been identified as a molecular driver of cancer in nearly 30% of all tumors and is heavily implicated in processes like metastasis. Previous research has suggested that Ras may function as homodimers, but the nature and role of this oligomerization in regulating Ras function is unclear. The single-cell eukaryote Dictyostelium discoideum has proven to be a great model to study Ras signaling and function, mostly due to its simpler signaling network. To investigate Ras dimerization in Dictyostelium, Bioluminescence Resonance Energy Transfer (BRET) was employed which allows for studying real-time protein-protein interactions in live cells. Using the novel GoldenBraid cloning method, the desired BRET constructs were generated by fusing the energy donor Renilla luciferase and energy acceptor GFP2 in-frame at the N-terminus of the RasG protein. Both BRET constructs were expressed in rasG null cells and the integrity of the fusion proteins and functionality of the reporters were verified. The BRET2 assay was performed on vegetative rasG null cells expressing GFP2-RasG and Rluc2-RasG and homodimerization was not observed. Future experiments will explore RasG homodimerization in chemotactically competent cells pulsed with cAMP.