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

Joshua J. Sims, Francesco Scavone, Eric M. Cooper, Lesley A. Kane, Richard J. Youle, Jef D. Boeke, Robert E. Cohen

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)303-309
Number of pages7
JournalNature Methods
Volume9
Issue number3
DOIs
StatePublished - Mar 1 2012

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Sims, J. J., Scavone, F., Cooper, E. M., Kane, L. A., Youle, R. J., Boeke, J. D., & Cohen, R. E. (2012). Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nature Methods, 9(3), 303-309. https://doi.org/10.1038/nmeth.1888