UC Berkeley

Cellular membranes organize signal transduction, serving as platforms for protein interactions, as well as direct modulators of enzyme function. Since many signaling reactions converge at the plasma membrane, understanding the coupling between protein

function and membrane localization of signaling complex is of fundamental importance. However, a quantitative description of many of such interactions has been lacking, largely due to the difficulty in performing measurements in the complex cellular environment. Here, we reconstitute and quantitatively probe the kinetics between small GTPase Ras and its activators: Son of Sevenless (SOS) and Ras guanyl nucleotide-releasing protein

(RasGRP) using supported membranes.

The activation of the membrane-associated Ras by cytosolic guanine nucleotide exchange factors, SOS and RasGRP, is a key step in a plethora of receptor-mediated cell signaling pathways. Both SOS and RasGRP contain multiple domains and several of which

are known to regulate the activation of SOS/RasGRP. In this dissertation, we study in parallel the kinetics of SOS and RasGRP-catalyzed Ras activation as well as the mechanisms that release the autoinhibition of SOS/RasGRP at single molecule level.

We develop an assay with supported bilayers functionalized with H-Ras to monitor the enzymatic activities from hundreds of individual SOS proteins. The data reveals that SOS is dynamically heterogeneous, sampling a broad distribution of turnover rates via

stochastic fluctuations between distinct states. The regulatory domains allosterically inhibit SOS by suppressing fluctuations to high activity states with sensitivity to both Ras nucleotide state and lipids. On the subject of Ras activation by RasGRP, the functional role of lipid interacting C1 domain of RasGRP1 has been studied. The preliminary results suggest that diacylglycerol (DAG), through the interaction with the C1 domain, regulates the function of RasGRP1 primarily by increasing its membrane localization.