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.
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Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).
Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).
Dikic, I., Wakatsuki, S. & Walters, K.J. Ubiquitin-binding domains—from structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 (2009).
Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. EMBO Rep. 9, 536–542 (2008).
Finley, D. et al. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol. Cell. Biol. 14, 5501–5509 (1994).
Spence, J., Sadis, S., Haas, A.L. & Finley, D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 15, 1265–1273 (1995).
Xu, M., Skaug, B., Zeng, W. & Chen, Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta. Mol. Cell 36, 302–314 (2009).
Pickart, C.M. & Cohen, R.E. Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 (2004).
Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).
Messick, T.E. & Greenberg, R.A. The ubiquitin landscape at DNA double-strand breaks. J. Cell Biol. 187, 319–326 (2009).
Kraft, C., Peter, M. & Hofmann, K. Selective autophagy: ubiquitin-mediated recognition and beyond. Nat. Cell Biol. 12, 836–841 (2010).
Lauwers, E., Erpapazoglou, Z., Haguenauer-Tsapis, R. & Andre, B. The ubiquitin code of yeast permease trafficking. Trends Cell Biol. 20, 196–204 (2010).
Liu, S. & Chen, Z.J. Expanding role of ubiquitination in NF-kappaB signaling. Cell Res. 21, 6–21 (2011).
Sims, J.J. & Cohen, R.E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80. Mol. Cell 33, 775–783 (2009).
Sims, J.J., Haririnia, A., Dickinson, B.C., Fushman, D. & Cohen, R.E. Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains. Nat. Struct. Mol. Biol. 16, 883–889 (2009).
Swanson, K.A., Kang, R.S., Stamenova, S.D., Hicke, L. & Radhakrishnan, I. Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J. 22, 4597–4606 (2003).
Kaiser, S.E. et al. Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools. Nat. Methods 8, 691–696 (2011).
Matsumoto, M.L. et al. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol. Cell 39, 477–484 (2010).
Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).
Geisler, S. et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119–131 (2010).
Narendra, D., Kane, L.A., Hauser, D.N., Fearnley, I.M. & Youle, R.J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy 6, 1090–1106 (2010).
Narendra, D., Tanaka, A., Suen, D.F. & Youle, R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008).
Arnason, T. & Ellison, M.J. Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Mol. Cell. Biol. 14, 7876–7883 (1994).
Skaug, B., Jiang, X. & Chen, Z.J. The role of ubiquitin in NF-kappaB regulatory pathways. Annu. Rev. Biochem. 78, 769–796 (2009).
Dynek, J.N. et al. c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J. 29, 4198–4209 (2010).
Saitoh, T. et al. TWEAK induces NF-kappaB2 p100 processing and long lasting NF-kappaB activation. J. Biol. Chem. 278, 36005–36012 (2003).
Feltham, R. et al. Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding. J. Biol. Chem. 285, 17525–17536 (2010).
Hjerpe, R. et al. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep. 10, 1250–1258 (2009).
Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117–120 (2004).
Wang, H. et al. Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. Proc. Natl. Acad. Sci. USA 105, 20197–20202 (2008).
Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).
Pickart, C.M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005).
Bremm, A., Freund, S.M. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010).
Millard, B.L., Niepel, M., Menden, M.P., Muhlich, J.L. & Sorger, P.K. Adaptive informatics for multifactorial and high-content biological data. Nat. Methods 8, 487–493 (2011).
Janes, K.A. et al. The response of human epithelial cells to TNF involves an inducible autocrine cascade. Cell 124, 1225–1239 (2006).
Janes, K.A. et al. A systems model of signaling identifies a molecular basis set for cytokine-induced apoptosis. Science 310, 1646–1653 (2005).
We thank S. Beese-Sims for constructing the leucine-auxotrophic version of the single-ubiquitin yeast strains, P. Sorger and B. Millard for helpful discussions about the manuscript and technical assistance on NF-κB activation experiments, A. Sliva for assistance with yeast experiments, B. Schmitt for assistance with binding experiments, A. Hess for help with statistical analyses and T. Yao for many helpful discussions. J.J.S. is supported by a fellowship from the Damon Runyon Cancer Research Foundation (DRG#2073-11). This work was supported, in part, by US National Institutes of Health (NIH) Common Fund grant RR020839 (J.D.B.), NIH grant P01 CA139980 (P. Sorger), US National Institute of Neurologial Disorders and Stroke Intramural Program (R.J.Y.), and NIH grants RC1 GM091424 (R.E.C.) and 1R01 GM097452 (R.E.C.).
A US patent application (12/815,74) describing design and applications of linkage-specific polyubiquitin binding proteins has been filed by Johns Hopkins University on behalf of J.J.S. and R.E.C.
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Sims, J., Scavone, F., Cooper, E. et al. Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat Methods 9, 303–309 (2012). https://doi.org/10.1038/nmeth.1888
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