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 2012

Fingerprint

Polyubiquitin
Cell signaling
Ubiquitin
Sensors
Proteins
Ubiquitins
Topology
Tumor Necrosis Factor Receptors
Interleukin-1
Proteomics
Yeast
Carrier Proteins
Yeasts
Chemical activation
Cells
Apoptosis
Imaging techniques
Substrates

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Biochemistry
  • Cell Biology

Cite this

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

Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. / Sims, Joshua J.; Scavone, Francesco; Cooper, Eric M.; Kane, Lesley A.; Youle, Richard J.; Boeke, Jef D.; Cohen, Robert E.

In: Nature Methods, Vol. 9, No. 3, 03.2012, p. 303-309.

Research output: Contribution to journalArticle

Sims, JJ, Scavone, F, Cooper, EM, Kane, LA, Youle, RJ, Boeke, JD & Cohen, RE 2012, 'Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling', Nature Methods, vol. 9, no. 3, pp. 303-309. https://doi.org/10.1038/nmeth.1888
Sims, Joshua J. ; Scavone, Francesco ; Cooper, Eric M. ; Kane, Lesley A. ; Youle, Richard J. ; Boeke, Jef D. ; Cohen, Robert E. / Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. In: Nature Methods. 2012 ; Vol. 9, No. 3. pp. 303-309.
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