Ubiquitin Chain Editing Revealed by Polyubiquitin Linkage-Specific Antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

[1]  Crafford A. Harris,et al.  Interleukin (IL)-1 Receptor–associated Kinase (IRAK) Requirement for Optimal Induction of Multiple IL-1 Signaling Pathways and IL-6 Production , 1998, The Journal of experimental medicine.

[2]  Steven P Gygi,et al.  Certain Pairs of Ubiquitin-conjugating Enzymes (E2s) and Ubiquitin-Protein Ligases (E3s) Synthesize Nondegradable Forked Ubiquitin Chains Containing All Possible Isopeptide Linkages* , 2007, Journal of Biological Chemistry.

[3]  P. Cohen,et al.  Interleukin-1 (IL-1) Induces the Lys63-Linked Polyubiquitination of IL-1 Receptor-Associated Kinase 1 To Facilitate NEMO Binding and the Activation of IκBα Kinase , 2008, Molecular and Cellular Biology.

[4]  C. Eigenbrot,et al.  High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. , 2004, Journal of molecular biology.

[5]  Matthew T Wheeler,et al.  The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses , 2004, Nature Immunology.

[6]  M. Carson,et al.  Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). , 1993, The Journal of biological chemistry.

[7]  S. Gygi,et al.  Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology , 2006, Nature Cell Biology.

[8]  Zhengfan Jiang,et al.  Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation* , 2008, Journal of Biological Chemistry.

[9]  J. Tschopp,et al.  Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation. , 2003, Immunity.

[10]  D. Fushman,et al.  Structural properties of polyubiquitin chains in solution. , 2002, Journal of molecular biology.

[11]  S. Ghosh,et al.  Shared Principles in NF-κB Signaling , 2008, Cell.

[12]  D. Wallach.  Faculty Opinions recommendation of De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. , 2004 .

[13]  Frederic A. Fellouse,et al.  High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. , 2007, Journal of molecular biology.

[14]  D. Ecker,et al.  Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant , 1994, Molecular and cellular biology.

[15]  J. Ashwell,et al.  Lys63-Linked Polyubiquitination of IRAK-1 Is Required for Interleukin-1 Receptor- and Toll-Like Receptor-Mediated NF-κB Activation , 2008, Molecular and Cellular Biology.

[16]  Zhijian J. Chen,et al.  Activation of the IκB Kinase Complex by TRAF6 Requires a Dimeric Ubiquitin-Conjugating Enzyme Complex and a Unique Polyubiquitin Chain , 2000, Cell.

[17]  Douglas K. Miller,et al.  The Interleukin-1 Receptor-associated Kinase Is Degraded by Proteasomes following Its Phosphorylation* , 1997, The Journal of Biological Chemistry.

[18]  S. Sidhu,et al.  Phage display for selection of novel binding peptides. , 2000, Methods in enzymology.

[19]  Steven P Gygi,et al.  A proteomics approach to understanding protein ubiquitination , 2003, Nature Biotechnology.

[20]  S. Akira,et al.  Essential function for the kinase TAK1 in innate and adaptive immune responses , 2005, Nature Immunology.

[21]  M. Kelliher,et al.  The kinase activity of Rip1 is not required for tumor necrosis factor-alpha-induced IkappaB kinase or p38 MAP kinase activation or for the ubiquitination of Rip1 by Traf2. , 2004, The Journal of biological chemistry.

[22]  S. Wasserman,et al.  Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. , 1999, Journal of immunology.

[23]  P. Cohen,et al.  The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1. , 2008, The Biochemical journal.

[24]  Somasekar Seshagiri,et al.  De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling , 2004, Nature.

[25]  Zhijian J. Chen,et al.  TAK1 is a ubiquitin-dependent kinase of MKK and IKK , 2001, Nature.

[26]  C. Pickart,et al.  Controlled synthesis of polyubiquitin chains. , 2005, Methods in enzymology.

[27]  Howard Schulman,et al.  Global changes to the ubiquitin system in Huntington's disease , 2007, Nature.

[28]  A. Haas,et al.  A ubiquitin mutant with specific defects in DNA repair and multiubiquitination , 1995, Molecular and cellular biology.

[29]  J. Tschopp,et al.  Recruitment of TNF Receptor 1 to Lipid Rafts Is Essential for TNFα-Mediated NF-κB Activation , 2003 .

[30]  Gabriel Pineda,et al.  Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. , 2006, Molecular cell.