A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta

Mol Cell. 2009 Oct 23;36(2):302-14. doi: 10.1016/j.molcel.2009.10.002.


Lysine-63 (K63)-linked polyubiquitination has emerged as a mechanism regulating diverse cellular functions, including activation of the protein kinase IKK in the NF-kappaB pathways. However, genetic evidence for a key role of K63 polyubiquitination in IKK activation is lacking. Here, we devise a tetracycline-inducible RNAi strategy to replace endogenous ubiquitin with a K63R mutant in a human cell line. We demonstrate that K63 of ubiquitin and the catalytic activity of Ubc13, an E2 that catalyzes K63 polyubiquitination, are required for IKK activation by IL-1beta, but surprisingly, not by TNFalpha. We further show that IKK activation by TNFalpha requires Ubc5, which functions with the E3 cIAP1 to catalyze polyubiquitination of RIP1 not restricted to K63 of ubiquitin. These results indicate that distinct ubiquitin-dependent mechanisms are employed for IKK activation by different pathways. The ubiquitin replacement methodology described here provides a means to investigate the function of polyubiquitin topology in various cellular processes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocatalysis / drug effects
  • Enzyme Activation / drug effects
  • Gene Knock-In Techniques
  • Genetic Techniques*
  • Humans
  • I-kappa B Kinase / metabolism*
  • Inhibitor of Apoptosis Proteins / metabolism
  • Interleukin-1beta / pharmacology*
  • Lysine / metabolism
  • Nuclear Pore Complex Proteins / metabolism
  • Polyubiquitin / metabolism
  • RNA-Binding Proteins / metabolism
  • Tetracycline / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Ubiquitin / genetics*
  • Ubiquitin / metabolism*
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitination / drug effects


  • AGFG1 protein, human
  • Inhibitor of Apoptosis Proteins
  • Interleukin-1beta
  • Nuclear Pore Complex Proteins
  • RNA-Binding Proteins
  • Tumor Necrosis Factor-alpha
  • Ubiquitin
  • Polyubiquitin
  • UBE2N protein, human
  • Ubiquitin-Conjugating Enzymes
  • I-kappa B Kinase
  • Tetracycline
  • Lysine