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Nat Methods. 2012 Feb 5;9(3):303-9. doi: 10.1038/nmeth.1888.

Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.

Author information

1
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.

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.

PMID:
22306808
PMCID:
PMC3438894
DOI:
10.1038/nmeth.1888
[Indexed for MEDLINE]
Free PMC Article

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