A Ubiquitin Replacement Strategy in Human Cells Reveals Distinct Mechanisms of IKK Activation by TNFα and IL-1β

Ming Xu, Brian Skaug, Wenwen Zeng, Zhijian J. Chen

Research output: Contribution to journalArticle

183 Scopus citations

Abstract

Lysine-63 (K63)-linked polyubiquitination has emerged as a mechanism regulating diverse cellular functions, including activation of the protein kinase IKK in the NF-κB 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-1β, but surprisingly, not by TNFα. We further show that IKK activation by TNFα 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.

Original languageEnglish (US)
Pages (from-to)302-314
Number of pages13
JournalMolecular cell
Volume36
Issue number2
DOIs
StatePublished - Oct 23 2009

Keywords

  • MOLIMMUNO
  • PROTEINS
  • SIGNALING

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'A Ubiquitin Replacement Strategy in Human Cells Reveals Distinct Mechanisms of IKK Activation by TNFα and IL-1β'. Together they form a unique fingerprint.

Cite this