Abstract

Ras GTPases are key regulators of cell growth, differentiation and transformation. Ubiquitination of Ras modulates its intracellular trafficking and signaling output. Yet, detailed knowledge about the in vivo dynamics of this process is limited. Here, we took advantage of the Bioluminescence Resonance Energy Transfer (BRET) technology to monitor HRas ubiquitination in living cells and to investigate its relationship to membrane compartmentalization. We demonstrate that HRas ubiquitination is dynamically altered upon growth factor stimulation. The growth factor‐dependent modulation of HRas ubiquitination is temporally coupled to changes in HRas microdomain localization as determined by BRET analysis of HRas/Caveolin1 interactions. Significantly, an HRas chimera that is unable to interact with Caveolin1 is refractory to ubiquitination, indicating that HRas ubiquitination requires an interaction with Caveolin1. Consistent with this observation, disruption of lipid microdomains by membrane cholesterol depletion inhibits HRas ubiquitination. Furthermore, Caveolin1 expression levels are tightly correlated with HRas ubiquitination. Taken together, our data demonstrate that HRas ubiquitination is dynamically regulated and requires the integrity of caveolae and their scaffolding protein Caveolin1.Supported by Human Frontier Science Program (S.T.) and NIH grant CA055360 (D.B.‐S.)

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