Biochemical and biophysical analyses of Ras modification by ubiquitin

Ras proteins are small GTPases that play key roles in the regulation of several cellular processes such as growth, differentiation, and transformation. Although Ras signaling was thought to occur uniformly on the inner leaflet of the plasma membrane, a growing body of evidence indicates that Ras activation happens dynamically within defined plasma membrane microdomains and at other specific intracellular compartments, thus ensuring the generation of distinct signal outputs. Yet the mechanisms that control the spatiotemporal segregation of Ras proteins remain poorly characterized. We have recently shown that the differential modification of Ras proteins by ubiquitination is a crucial factor that controls Ras intracellular trafficking and signaling potential. To better understand the process of Ras ubiquitination, it is important to establish assays that not only provide information about the nature of the ubiquitin modification involved, but also enable the monitoring of the dynamics of this process. In this chapter, we will describe biochemical and biophysical methodologies, namely immunoprecipitation, nickel-chelate affinity chromatography, and bioluminescence resonance energy transfer (BRET), for monitoring the ubiquitination of Ras proteins. Although the description focuses on Ras, the assays described can in principle be applied to the study of a range of proteins of interest that may be subject to ubiquitination, and the use of the different methods in parallel should provide new insights into the nature and dynamics of protein ubiquitination.