Ubiquitin Telling Polys Apart

John F. Foley

Science Signaling, AAAS, Washington, DC 20005, USA

Polyubiquitination is a tightly regulated posttranslational modification of proteins that has important implications for their function. When individual ubiquitin molecules in a chain are linked to each other through Lys⁴⁸ (K48) residues, the ubiquitinated protein is often targeted for proteasomal degradation. However, if the linkages occur through Lys⁶³ (K63), the ubiquitinated protein is not degraded; instead, its activity is often enhanced. Distinguishing between these polyubiquitin chains has been technically challenging and often involves mass spectrometry or the use of mutant ubiquitin molecules. By screening a phage display library for low-affinity antibodies against specific polyubiquitin chains, solving the structures of some of the positive clones, and then using these structures to design changes to engineered antibodies, Newton et al. developed linkage-specific antibodies that recognized either K48- or K63-linked polyubiquitinated proteins. Immunofluorescent and immunohistochemical analyses performed with these antibodies showed, as expected, that K48-linked, but not K63-linked, polyubiquitinated proteins colocalized with proteasomes. Stimulation of the tumor necrosis factor receptor (TNFR) is known to lead to the recruitment and K63-linked polyubiquitination of the adaptor kinase RIP1, which enables RIP1 to activate nuclear factor B (NF-B) signaling. The authors first used their antibodies to confirm the K63-linked polyubiquitination of RIP1 immediately after TNFR stimulation and then found that RIP1 was later polyubiquitinated by K48-linked chains and degraded, coincident with the termination of NF-B signaling. A similar pattern of polyubiquitin editing was found for interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1), an adaptor kinase recruited to activated Toll-like receptors. These antibodies will likely prove very useful in determining whether other adaptor kinases undergo polyubiquitin editing to modulate their function and in identifying other modified proteins.

K. Newton, M. L. Matsumoto, I. E. Wertz, D. S. Kirkpatrick, J. R. Lill, J. Tan, D. Dugger, N. Gordon, S. S. Sidhu, F. A. Fellouse, L. Komuves, D. M. French, R. E. Ferrando, C. Lam, D. Compaan, C. Yu, I. Bosanac, S. G. Hymowitz, R. F. Kelley, V. M. Dixit, Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668-678 (2008). [Online Journal]

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