Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.
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.
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.
Joshua J
JJ
Sims
Francesco
F
Scavone
Eric M
EM
Cooper
Lesley A
LA
Kane
Richard J
RJ
Youle
Jef D
JD
Boeke
Robert E
RE
Cohen
D000818:Animals; D001665:Binding Sites; D006801:Humans; D015336:Molecular Probe Techniques; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*
D025801:Ubiquitin