A study of the membrane interactions of K-ras

Lau, Chiyan (2006). A study of the membrane interactions of K-ras PhD Thesis, Institute for Molecular Bioscience , University of Queensland.

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Author Lau, Chiyan
Thesis Title A study of the membrane interactions of K-ras
School, Centre or Institute Institute for Molecular Bioscience
Institution University of Queensland
Publication date 2006
Thesis type PhD Thesis
Supervisor Prof John Hancock
Abstract/Summary Ras proteins are monomeric GTPases which operate in cellular signal transduction pathways to regulate cell growth, differentiation and apoptosis. Mutations at amino acid residue 12, 13 or 61 render the protein constitutively active and oncogenic. These mutations are found in 30% of human cancers. There are three isoforms of the Ras protein: H-ras, N-ras and K-ras. All three isoforms localize to the inner leaflet of the plasma membrane, and this localization is important for their biological function. H-ras and N-ras traffic to the plasma membrane via the classical exocytic pathway, while the K-ras trafficking pathway is non-vesicular and is currently poorly characterized. In order to identify the proteins that may be involved in the K-ras trafficking pathway, an affinity chromatography approach was developed. The method utilized the C-terminal membrane-targeting regions of K-ras (CTK) and H-ras (CTH) as baits to purify cellular proteins from mouse brain cytosol. Proteins which selectively interacted with CTK but not CTH were identified by mass spectrometry. Four of these proteins, B23, Ncl, LANCL1 and laminin receptor-1, were further characterized and evaluated as putative K-ras transport factors. B23 localizes to P100 and nuclear fractions, and selectively interacts with K-ras in vivo. However, overexpression or siRNA-mediated knockdown of B23 does not affect plasma membrane localization of K-ras. Ncl localizes to the P100, S100 and nuclear fractions. Nuclear Ncl, but not extranuclear Ncl, selectively interacts with K-ras in vivo. Overexpression of Ncl does not affect the intracellular localization of either H-ras or K-ras. LANCL1 is associated with the P100, S100 and nuclear fractions, and preliminary results show that LANCL1 may selectively interact with K-ras in vivo. Selective interaction between laminin receptor-1 and K-ras could not be confirmed, either in vitro or in vivo. Also, overexpression of laminin receptor-1 has no effect on the P100/S100 distribution of K-ras, or its downstream MEK/ERK signalling. In conclusion, this thesis represents the first known proteomic approach dedicated to the discovery of K-ras transport factors. Three novel selective interacting partners of the membrane targeting domain of K-ras are identified, although their precise function in K-ras trafficking remains to be elucidated.

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