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Mechanisms of Ras Signaling and Activation on Reconstituted Membrane Surfaces


The membrane is a crucial component of the cell that modulates protein spatial organization and enzyme-substrate reactions, which, in turn, mediates cellular signal transmission. One key membrane protein is the small GTPase Ras, which acts as a signaling node that determines cellular outcome. Though Ras is an attractive target for anticancer therapies, effective treatments against its oncogenic mutants remain elusive due to the incomplete understanding of its signaling potential in the membrane environment. Here, we seek to elucidate some of the key biophysical properties and regulations of Ras on membranes in reconstituted systems.

Lateral organization, especially dimerization, of Ras on cellular membranes has long been a subject of great interest. We find that Ras dimers can form on membranes by photosensitization reactions, in which molecular oxygen mediates protein radicalization under typical fluorescence experimental conditions. Inter-protein dityrosine crosslinking is one of the dimerization motifs, and the specific surface tyrosine distribution on Ras renders the protein especially sensitive to this reaction. Photosensitization reactions are reflective of physiological oxidative stress induced by reactive oxygen species, suggesting such processes may occur naturally in cells.

Ras activation is a critical step for receptor tyrosine kinase signal transduction. One key activator of Ras is the enzyme Son of Sevenless (SOS), which catalyzes Ras nucleotide exchange. The complex domain architecture of SOS ensures efficient Ras turnover only when all activation inputs are met, including stable membrane recruitment. Notably, a single molecule of SOS can processively turnover thousands of Ras molecules upon allosteric Ras engagement, and generation of Ras.GTP creates positive feedback by enhancing SOS recruitment. We developed a real-time Ras activation assay to detect the activity of purified, full-length SOS (SOSFL) molecules exposed to inactive Ras on membranes. Despite the collective autoinhibition from the N- and C-terminal domains of the enzyme, SOSFL is nonetheless capable of inducing rapid Ras activation by single proteins. Our measurements further reveal a new mechanism of Ras.GTP-mediated SOS activation through enhancement in probability of single SOS to transition to the hyperactive state. Processively active single SOS molecules are thus consequential in Ras signaling and additional measures are necessary to suppress spurious Ras activation.

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