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Function and Regulation of RhoBTB1

Student thesis: Doctoral ThesisDoctor of Philosophy

Rho GTPases are a family of proteins known to be involved in cytoskeletal regulation and are important for several processes including cell migration, cell polarity, vesicle trafficking and cytokinesis. RhoBTB1 is an atypical member of the Rho GTPase family. It consists of a non-functional GTP-binding domain followed by a proline-rich region and two tandem BTB domains. The only known interacting partner for RhoBTB1 is cullin3, a scaffold protein in ubiquitin ligase complexes. So far RhoBTB1 has not been shown to affect the cytoskeleton and it has no known cellular function. Most Rho GTPases are regulated by GEFs, GAPs, RhoGDIs and post-translational lipid modifications at the C-terminus. However, RhoBTB1 is not regulated by any of these mechanisms. RhoBTB1 has additional domains that could be involved in protein-protein interaction, leading to an alternative mechanism for RhoBTB1 regulation. This project has shown that RhoBTB1 can interact with RhoA and ROCK1 as well as cullin3. The interaction between RhoA and RhoBTB1 was explored since RhoBTB1 has the potential to recruit substrates for ubiquitination by cullin3 complexes. The region of interaction between RhoA and RhoBTB1 was mapped and RhoBTB1 influenced the protein level of RhoA, suggesting that it inhibits RhoA degradation by the proteasome. RhoBTB1 was found to localise diffusely in the cytoplasm or to punctate structures. Knockdown of RhoBTB1 led to a change in cell morphology in a 3D Matrigel matrix, indicating that it influences cell shape in 3D, although it did not alter cell shape on 2D substrata. RhoBTB1 can be phosphorylated by ROCK1 in vitro and the region of interaction between RhoBTB1 and ROCK1 was mapped using ROCK1 deletion mutants. I hypothesize that RhoBTB1 interacts with RhoA to affect its ubiquitination and degradation and hence affects cell morphology in a 3D matrix, and that RhoBTB1 activity is regulated by ROCK1-mediated phosphorylation.
Original languageEnglish
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Award date2016

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