Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin–binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1β and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.
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We thank S. Beese-Sims for constructing the leucine-auxotrophic version of the single-ubiquitin yeast strains, P. Sorger and B. Millard for helpful discussions about the manuscript and technical assistance on NF-κB activation experiments, A. Sliva for assistance with yeast experiments, B. Schmitt for assistance with binding experiments, A. Hess for help with statistical analyses and T. Yao for many helpful discussions. J.J.S. is supported by a fellowship from the Damon Runyon Cancer Research Foundation (DRG#2073-11). This work was supported, in part, by US National Institutes of Health (NIH) Common Fund grant RR020839 (J.D.B.), NIH grant P01 CA139980 (P. Sorger), US National Institute of Neurologial Disorders and Stroke Intramural Program (R.J.Y.), and NIH grants RC1 GM091424 (R.E.C.) and 1R01 GM097452 (R.E.C.).
A US patent application (12/815,74) describing design and applications of linkage-specific polyubiquitin binding proteins has been filed by Johns Hopkins University on behalf of J.J.S. and R.E.C.
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Sims, J., Scavone, F., Cooper, E. et al. Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat Methods 9, 303–309 (2012). https://doi.org/10.1038/nmeth.1888
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