Compartmentalized signalling of Ras in cancer cells
Final Report Abstract
En route to the their final destination at the Plasma membrane (PM) Ras proteins experience a series of post-translational modifications, notably the attachment of palmitate fatty acid groups, which determine the subcellular trafficking and residency of the Ras holoproteins. The subcellular segregation of Ras to distinct membrane compartments including the PM and endomembranes is considered a potentially important means to enable signal diversification in cellular signal transduction. Since this principle can in theory also modulate the subcellular distribution of oncogenic Ras, we originally devised this study in order to investigate the possibility that oncogenic Ras activity was restricted to specific subcellular platforms that differed from the sites of native Ras signalling. Owing to an unexpectedly strong addiction and dependence of the Ras-transformed cells to the Rasoncoprotein, we were unable to accomplish this task because our fluorescent biosensors for active Ras-GTP are at the same time potent scavengers of Ras activity. Instead we designed a series of experimental approaches that have enabled us to manipulate the subcellular distribution of Ras proteins and to test the role of the location in the ability of Ras to respond to growth factors and to transmit their signals. Amongst other methodologies, we have visualized the activation of Ras in live MCF-7 cells, a breast cancer cell line that features Ras gene amplification. The experimental data obtained by use of all approaches, which further included the use of palmitoylation-deficient Ras mutants and the determination of the palmitoylation status of active Ras provided an unexpected, unambiguous result: Ras proteins (specifically: the palmitolyated Ras species N-Ras and H-Ras) feature an absolute requirement for palmitoylation in order to exert their biological activity as signal mediators in hormonal signalling. Since the subcellular distribution of Ras is largely dictated by its palmitoylation status, our data further illustrate that Ras must reside at the PM to become engaged by growth factor receptors. These are striking and far-reaching observations since they largely exclude a role for endomembranes as a platform for Ras signalling in the context of growth factor action. This conclusion can also be applied to MCF-7 cells, evidencing that at least in this scenario of cellular transformation, aberrant Ras signalling will originate at the PM. However, due to the unexpected experimental drawbacks mentioned above we can, at this time point, not extrapolate this notion to other settings of Ras-dependent transformation. In conclusion, our findings document that Rasactivation and Ras-signal transmission require palmitoylation of the Ras protein and proceed at the PM. Importantly, the strict requirement for palmitoylation raises the possibility of blocking this processing step as a new means of interference with oncogenic Ras-driven transformation.
Publications
- “Life cell imaging of endogenous Ras activation by the T cell receptor”, in “Joint meeting der Signal Transduction Society (STS)”, Weimar, 28.10.2009 - 30.10.2009
- (2010) Ras activation by the T-cell receptor proceeds at the plasma membrane and requires palmitoylation of N-Ras. J. Immunology 15 (185):3536-43
I. Rubio, S. Grund, S. Song, C. Biskup, S. Bandemer, M. Fricke, M. Förster, A. Graziani, U. Wittig, S. Kliche
- “Role of lipid modifications in the sub-cellular localization and activation of Ras”, 15.3.2011, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, UK
