Investigation of Ras trafficking and plasma membrane interaction

Kwang-jin Cho (2009). Investigation of Ras trafficking and plasma membrane interaction PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Kwang-jin Cho
Thesis Title Investigation of Ras trafficking and plasma membrane interaction
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2009-02
Thesis type PhD Thesis
Supervisor Prof. John F Hancock
Prof. Rob G Parton
Dr. Michelle Hill
Total pages 157
Total colour pages 67
Total black and white pages 90
Subjects 06 Biological Sciences
Abstract/Summary Ras GTPases are crucial regulators of cellular signalling that function as molecular switches and regulate important cellular processes such as differentiation, proliferation, and apoptosis. Mutations in ras genes that render Ras GTPases permanently active have been known for over 25 years to be crucial in tumour development and progression. Approximately 15% of all human cancers have activating point mutations in ras genes, and different Ras isoforms contribute to the development of different types of cancer. The development of drugs that effectively interfere with Ras signalling and possibly a drug that is isoform-specific, therefore remain of the highest importance. In this thesis, I present a screening assay I have developed to discover microbial compounds that disrupt the plasma membrane localisation of Ras GTPases. The assay allows to discover compounds targeting proteins involved in trafficking and/or plasma membrane localisation of Ras. Its unique design led to the discovery of seven microbial extracts and two pure compounds (staurosporine-derivatives and bafilomycin) that mislocalise K- and/or H-Ras. Identification of the active compounds and their mechanisms of action is an excellent opportunity to uncover the yet unknown trafficking pathway of K-Ras, and it is an important first step towards the development of K-Ras-specific anti-cancer drugs. Statins are HMG-CoA reductase inhibitors that are used to reduce cholesterol levels in hypercholesterolemia patients. In addition to their cholesterol-lowering effect, extensive studies have shown pleiotropic effects of the drug, which are suggested to be cholesterol-independent. In particular, it has been proposed that at least some of these pleiotropic effects are due to inhibition of prenylation of Rho and Ras proteins, which would prevent their correct function. However, this hypothesis has never been proven in patients or when the drug is used at therapeutic levels. I will demonstrate that clinical doses of lovastatin do not prevent protein prenylation and that cholesterol depletion by lovastatin actually stimulates, rather than inhibits, the activities of Ras and MAPK via phospholipase D2. Statins may also interact with proteins other than HMG-CoA reductase. In complementary experiments I therefore designed a novel lovastatin pulldown and discovered a direct interaction of lovastatin with protease-activated receptor 2, a G-protein-coupled receptor. I will demonstrate that lovastatin inhibits PAR-2 mediated intracellular calcium release. Thus, I suggest that statins may also alter cellular signalling by regulating the activity of signalling receptors on the plasma membrane. The pleiotropic effects of statins could therefore be a result of the existence of more than one target. In conclusion, I have shown that mislocalisation of Ras GTPases from the plasma membrane or disruption of the plasma membrane structure affects Ras signal transduction. These results re-confirm that the plasma membrane is an important platform for Ras GTPase signalling. Therefore, detailed characterisation of plasma membrane microdomains and their interactions with signalling proteins will provide new insights into cellular signalling, which could be a starting point for a new era of cancer therapies.
Keyword Ras GTPase
Signal transduction
Ras trafficking
Screening assay
Bafilomycin
Staurosporine
Plasma membrane
Lipid raft
HMG-CoA reductase inhibitor
phospholipase D2
Protease-activated Receptor 2
Additional Notes Colour pages: 1, 18, 20, 24-26, 28-29, 32, 33-34, 36, 38-39, 59, 61-69, 71-72, 74, 76-81, 83-84, 86-87, 91, 94, 96-97, 99, 101-102, 104-106, 108-114, 116, 123-126, 128-134, 141

 
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Created: Fri, 29 May 2009, 11:31:07 EST by Mr Kwang-jin Cho on behalf of Library - Information Access Service