Real-time monitoring of ubiquitination in living cells by BRET.
Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.
Perroy
Julie
J
Département de Biochimie and Groupe de Recherche sur le Système Nerveux Autonome, Université de Montréal, C.P. 6128 Succursale Centre-Ville Montréal, Québec H3C 3J7, Canada.
Pontier
Stephanie
S
Charest
Pascale G
PG
Aubry
Muriel
M
Bouvier
Michel
M
D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D008657:Metabolic Clearance Rate; D025941:Protein Interaction Mapping / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D014452:Ubiquitins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins