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- Title
Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.
- Authors
Sims, Joshua J; Scavone, Francesco; Cooper, Eric M; Kane, Lesley A; Youle, Richard J; Boeke, Jef D; Cohen, Robert E
- Abstract
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.
- Publication
Nature methods, 2012, Vol 9, Issue 3, p303
- ISSN
1548-7105
- Publication type
Journal Article
- DOI
10.1038/nmeth.1888