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

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.

A single NFκB system for both canonical and non-canonical signaling

“…Emerging evidence indicates that three possible pathways for IKK2 activation are utilized to varying degrees by different inflammatory receptors (Figure 3). In the case of the TNF receptor I, associated proteins recruit the E2/E3 ligase complex consisting of UbcH5 and cIAP1, which subsequently forms ubiquitin chains of various linkages to RIP1 [11,12]. The TAB/TAK1 and IKK complexes are able to bind these ubiquitin chains, allowing the activated TAK1 to phosphorylate and activate IKK2 [13].…”

“…The TAB/TAK1 and IKK complexes are able to bind these ubiquitin chains, allowing the activated TAK1 to phosphorylate and activate IKK2 [13]. Additionally, oligomerization of the NEMO-IKK2 complex upon mixed ubiquitin chain binding can allow for TAK1-independent trans-autophosphorylation and activation of the IKK2 complex [12]. Another pathway of IKK2 activation revolves around linear ubiquitin chain formation.…”

“…In the third pathway of IKK2 activation, ligand engagement of the IL-1R and TLRs leads to the recruitment of the E2/E3 ligase complex made up of Ubc13 and TRAF6 [16]. This complex conjugates K63-linked ubiquitin chains to IRAK1, allowing for TAK1 and IKK2 complex binding and subsequent TAK1 activation of IKK2 through phosphorylation [12]. While a molecular description of these three pathways has advanced, the stimulus specificity of each pathway remains to be delineated.…”

Small-Molecule Chemical Probe Rescues Cells from Mono-ADP-Ribosyltransferase ARTD10/PARP10-Induced Apoptosis and Sensitizes Cancer Cells to DNA Damage

“…The UIMs of ARTD10 have been shown to specifically interact with K63-linked poly-ubiquitin (K63-pUb) chains . K63-pUB chains function as scaffolds in various signaling processes including the NF-B signal transduction pathway (Chen and Sun, 2009;Fan et al, 2011;Xu et al, 2009). Indeed ARTD10 regulates NF-B signaling, where both its ADP-ribosylation activity and its interaction with K63-pUb by the UIMs are needed .…”

Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10

“…For example, K48-pUb marks proteins for proteasomal degradation. In contrast K63-pUb functions as a scaffold in signalling processes, including the nuclear factor-kB (NF-kB) signal transduction pathway [14][15][16] . NF-kB controls many key processes, such as proliferation and cell survival, and is important in regulating inflammation and tumorigenesis among others 17,18 .…”