A wealth of evidence supports the key role of ubiquitination in regulating signal transduction in the NF-κB pathways. It is now clear that ubiquitination not only controls the degradation of IκBs and the processing of NF-κB precursors (p100) by the proteasome, but also regulates the activation of IKK through proteasome-independent mechanisms. It is remarkable to note that many proteins involved in the regulation of IKK have ubiquitin-related functions, including ubiquitin conjugation (e.g., TRAFs, cIAPs and Ubcs), deconjugation (e.g., A20 and CYLD) and recognition (e.g., NEMO and TAB2). Ubiquitination also regulates the activation of MAP kinases (e.g., TAK1, JNK and p38) and TBK1, suggesting that ubiquitin has a more general role in regulating protein kinase activation in immune and inflammatory pathways.
The basic mechanism underlying ubiquitin signaling involves the interaction between ubiquitinated targets and ubiquitin receptors that harbor UBDs. This mechanism applies to both signaling to the proteasome and proteasome-independent functions of ubiquitin. The 19S regulatory particle of the proteasome contains several ubiquitin receptor subunits that recognize polyubiquitinated proteins destined for degradation 137
. As cells contain numerous proteins that are ubiquitinated and perhaps even more proteins that contain UBDs, how is a ubiquitinated protein sorted out and targeted to the proteasome or other destinations to perform specific regulatory functions? It is generally believed that different polyubiquitin chains on a protein target determine whether a protein is degraded by the proteasome or not. For example, K48 polyubiquitin chains target proteasomal degradation, whereas K63 polyubiquitination performs non-proteolytic functions. In support of this model, quantitative mass spectrometry shows that inhibition of the proteasome in yeast cells leads to the accumulation of ubiquitin chains of different linkages, except K63 linkage 138
. However, in vitro
experiments show that K63 polyubiquitinated proteins are efficiently degraded by the 26S proteasome 139
. The inefficient degradation of K63 polyubiquitinated proteins in vivo
may be due to their sequestration by other ubiquitin-binding proteins in cells, as well as their faster deconjugation by the proteasome-associated DUBs 140
In general, UBDs bind to ubiquitin and ubiquitin chains with low affinity and selectivity. Thus, the specificity of sorting different ubiquitinated proteins for different fates cannot rely on the sole interaction between ubiquitin and UBDs. Additional interactions between other domains of the ubiquitinated proteins and ubiquitin receptors can greatly enhance the specificity. Space and time must also play a key role in determining the specificity of ubiquitin signaling. Since ubiquitin chains are very labile due to abundant DUBs in cells, the “life time” of a ubiquitin chain on a given substrate is likely to be quite short. Thus, the interaction between a ubiquitinated protein and a ubiquitin receptor must occur in the right place at the right time. New technologies and approaches, such as high-resolution live-cell imaging and systems biology, should yield new insights into how space and time control the specificity of ubiquitin signaling.
Intense studies in the past few years on NF-κB signaling have not only uncovered the central role of ubiquitination in the regulation of IKK in different pathways, but also revealed an unexpected complexity of signaling mechanisms in these pathways. While there is strong evidence that K63 polyubiquitination is essential for IKK activation in the IL-1β pathway, the mechanism of IKK activation in the TNFα pathway appears to be more complex. Not only are Ubc13 and K63 polyubiquitination dispensable for IKK activation by TNFα, but also deletion of signaling proteins such as TRAF2, TRAF5, RIP1 or TAK1 only leads to a partial inhibition of IKK. The activated TNF receptor complex contains many proteins, including multiple ubiquitin ligases. Thus, it is possible that there is a considerable degree of redundancy in TNF signaling. For instance, K63 and other types of polyubiquitination, including linear and perhaps “mixed” polyubiquitination, may play redundant roles in IKK activation by TNFα. Unlike the IL-1 pathway in which IKK activation by TRAF6 can be reconstituted in cell-free extracts, it has not been possible to establish a cell-free system that mimics the TNFα pathway using proteins such as TRAF2, cIAP1 or HOIP. In-depth biochemical and genetic studies are required to unravel the complexity of IKK regulation by TNFα.
A common and important issue in ubiquitin research is the identification and validation of physiologically relevant ubiquitination targets. Normally, a very small fraction of a protein target is modified by ubiquitin, making it difficult to study the activity of the modified proteins and determine the modification sites. Rapid advances in mass spectrometry should help to overcome this technical hurdle. The validation of ubiquitination targets often relies on mutagenesis of the putative or confirmed ubiquitination sites. However, such mutagenesis data must be interpreted with caution, because mutations of certain residues can lead to unintended consequences, such as affecting ubiquitin binding, enzymatic functions or protein conformations. The recent finding that unanchored polyubiquitin chains have direct signaling functions in regulating the activity of TAK1, IKK and RIG-I adds another layer of complexity to ubiquitin signaling. A burning issue in NF-κB research is to sort out the role of many proposed ubiquitination targets (e.g., NEMO, TAK1, TAB2, RIPs, IRAKs, TRAFs, MALT1, BCL10, ELKS, RIG-I and TBK1) as well as unanchored polyubiquitin chains in IKK activation by different pathways. Clarifying this issue will also help to understand how DUBs such as A20 and CYLD inhibit IKK.