The regulation of intracellular signals allows immune cells to integrate stimuli from their environment and to exhibit the dynamic plasticity characteristic of immune responses. The transduction of such signals requires rapid post-translational modifications of proteins via processes such as phosphorylation or ubiquitination. While phosphorylation events are generally binary, comprised of the presence or absence of a single phosphate group on selected amino acids of target proteins (e.g., serine, threonine, or tyrosine), ubiquitination events include the attachment of a variety of lengths and conformations of ubiquitin chains, mostly on lysine residues (Pickart and Fushman, 2004
) Understanding how ubiquitination events are regulated, and how they regulate a diverse array of cellular responses () requires an understanding of the components of the ubiquitin system.
E3 ubiquitin ligases are intergral mediators of immune regulation.
Ubiquitination involves a three step enzymatic reaction catalyzed by three different types of proteins, termed E1, E2, and E3 ubiquitin ligases (Pickart and Eddins, 2004
). An E1 enzyme first “activates” ubiquitin by forming a thiol ester bond. Activated ubiquitin is transferred to an E2 ubiquitin conjugating enzyme. The E2- ubiquitin (Ub) interacts with an E3 ubiquitin ligase that facilitates transfer of the ubiquitin to the epsilon-amino group of a lysine (K) on substrate proteins. Together with ubiquitin binding proteins (or sensors) and proteases that function as de-ubiquitinating enzymes (DUBs), E1, E2, and E3 ubiquitin ligase complexes constitute the core biochemical machinery for building, editing and removing ubiquitin chains.
While two known E1 enzymes “charge” or activate ubiquitin molecules for virtually all ubiquitination events in the mammalian proteome, diverse combinations of E2 and E3 ubiquitin ligases attach distinct types of ubiquitin chains to specific substrate proteins. Approximately 38 E2 enzymes are predicted to exist (Ye and Rape, 2009
). As there are many more E3 ubiquitin ligases (>600 predicted) than E2s, and most E2s functionally interact with many E3 ubiquitin ligases. In addition, at least some E3s can bind to multiple E2s. For example, the E3 ubiquitin ligase complex of BRCA1 and BARD1 can interact with 10 different E2s that display divergent functions; one E2 may effect ubiquitin initiation and other E2s may effect the elongation of various linkages depending on the E2 used (Christensen et al., 2007
; Christensen and Klevit, 2009
). Hence, a vast number of E2 and E3 combinations are available to specify the target proteins to be modified and the type of ubiquitin chains to be added.
E2 enzymes play a major role in determining the length and linkage type of ubiquitin chains that are formed (reviewed in Christensen and Kelvit, 2009
; Ye and Rape, 2009
). For example, the E2 enzyme Ubc13 preferentially builds K63-linked ubiquitin chains that support mitogen activated protein (MAP) kinase signal propagation (Yamamoto et al, 2006
, Rodrigo-Brenni et al., 2010
; David et al., 2010
). Ubc13 is required for interleukin-1 (IL-1) and lipopolysaccharide (LPS) induced MAP kinase activation, but appears less important for Nuclear Factor-κB (NF-kB) signaling from these ligands in macrophages and fibroblasts (Yamamoto et al, 2006a
). Ubc13 also appears to be dispensable for tumor necrosis factor (TNF) induced NF-kB signaling. In contrast, Ubc13 is important for T cell receptor (TCR) induced NF-kB signaling in thymocytes (Yamamoto et al., 2006b
). It is possible that other E2 ligases, such as Ubc5, can support K63 ubiquitin dependent signals, depending on the cell type and stimulus. The selectivity of E2s for certain subsets of E3 enzymes (and hence substrates) and the predilection of E2s to form particular ubiquitin chain linkages combine to render these enzymes important regulators as well as mediators of ubiquitination.
E3 ubiquitin ligases confer substrate specificity to the ubiquitin reaction by binding to and mediating transfer of ubiquitin from E2 enzymes to target proteins such as signaling molecules. E3 ubiquitin ligases have been divided into two general types depending on the type of protein domain used to recognize substrates: R
ene (RING) and H
omologous to E
6-associated protein c
erminus (HECT) E3 ubiquitin ligases. RING E3s make up the largest number by far, with over 600 predicted to be encoded in the human genome, while 28 HECT E3s are predicted to exist (Deshaies et al., 2009
; Rotin and Kumar, 2009
RING and HECT E3s mediate substrate ubiquitination by different mechanisms. RING E3s use their RING finger domain to direct the transfer of ubiquitin from the activated E2-Ub to the substrate, whereas HECT E3s accept the ubiquitin from the E2-Ub to form a covalent thioester bond intermediate before transferring ubiquitin to the target protein. Because HECT E3s are charged with a ubiquitin while bound to their target protein, they may also help determine the specificity of linkage chain formation (Ye and Rape, 2009
). In addition, many RING E3s have ubiquitin binding domains (UBD) that may serve to orient the acceptor ubiquitin molecule for attack and thereby influence the type of linkage formed. Indeed, a subset of E3 ubiquitin ligases, occasionally termed E4 ligases, may preferentially extend ubiquitin chains on ubiquitinated substrates. Hence, E3 enzymes mediate target recognition and can also contribute to linkage specificity.
In addition to RING and HECT domain containing E3 ubiquitin ligases, other protein motifs, such as plant homology domains (PHD), U box domains, and a subset of zinc fingers, have been implicated in E3 ubiquitin ligase activity. The U box, so designated by the domain found in the yeast ubiquitination factor UFD2, is a modified RING finger that lacks the canonical cysteine residues for zinc binding but which can nevertheless mediate ubiquitin ligase activity (Aravind and Koonin, 2000
; Hatakeyama et al., 2001
). More recently, a novel zinc finger motif in the ubiquitin ligase A20 protein has been shown to mediate E3 ubiquitin ligase activity (Wertz et al, 2004
). Although all proteins bearing such motifs have not been tested for E3 ubiquitin ligase activity, it is likely that the number of bona fide E3 ubiquitin ligases will grow significantly. Moreover, E3 ubiquitin ligases typically possess the ability to ubiquitinate multiple substrates, suggesting that a significant portion of the mammalian proteome undergoes ubiquitination.
Ubiquitin chains of diverse conformations regulate immune signals
Ubiquitination events in immune cells mediate diverse cell signals and cellular responses. Part of this diversity is due to the fact that ubiquitin molecules can be attached to proteins as monomers or as polymers (). Mono-ubiquitination events regulate DNA repair, receptor endocytosis, vesicle sorting, gene silencing (Sigismund et al., 2004
). Ubiquitination of DNA repair proteins can impact immune processes such as class switch recombination (Li et al, 2010
; Santos et al, 2010
; Sun and Chen, 2004
). Monoubiquitination has also been implicated in persistent NF-kB signaling that has implications for human T cell leukemia virus-1 (HTLV-1) infection and signaling thresholds mediating positive and negative thymic selection (Carter et al. 2005
, Wada et al., 2009
; Wang et al., 2010
A diverse array of ubiquitin chain linkages can lead to different outcomes for the substrate protein and resulting in different cellular responses
Polyubiquitin chains can be formed using any one of the seven internal lysine residues (K6, K11, K27, K29, K33, K48, and K63) or the N-terminal amino group of ubiquitin (Komander, 2009
) to form distinct ubiquitin chain linkage types (). Structural studies have revealed that different chain linkages adopt distinct conformations (Pickart and Fushman, 2004
; Fushman and Walker, 2010
). Hence, distinct chain types could be distinguished by ubiquitin dependent proteins.
K48-linked ubiquitin chains that are at least four ubiquitin molecules in length target misfolded or senescent proteins for recruitment to the proteasome for proteolytic degradation (Pickart and Fushman, 2004
). In the context of cell signaling, K48-linked polyubiquitin chains facilitate degradation of signaling proteins, including both agonists and inhibitors of signal transduction. Signaling proteins probably do not exhibit the same biochemical features as misfolded proteins. E3 ubiquitin ligases that target signaling proteins often recognize modifications to these proteins that occur during their activation, such as phosphorylated residues. For example, phosphorylation of the NF-kB inhibitor IkBα leads to its recognition by a Skp1-Cul1-F box E3 complex called SCFβTrCP
that adds K48 ubiquitin chains to IkBα and promotes its degradation (Skaug et al., 2009
). Hence, regulated degradation of signaling inhibitors supports the propagation of canonical NF-kB signals. By contrast, K48 ubiquitination of agonist signaling proteins limits the duration of signals. An example of this type of ubiquitin regulation is the negative feedback inhibition of cytokine signaling by suppressors of cytokine signaling (SOCS) family proteins. SOCS proteins are E3 ubiquitin ligases that tag cytokine signaling proteins, such as Janus kinases (JAKs) and cytokine receptors, with ubiquitin, marking them for degradation (Alexander and Hilton, 2004
). Regulated attachment of K48 ubiquitin chains to agonist signaling molecules has been increasingly recognized in restricting various immune signaling cascades.
Alternative (non-K48) ubiquitin chains can target modified proteins for nonproteolytic functions
A major revelation in cell signaling biology was the discovery that ubiquitin chains assembled in certain conformations can target proteins for outcomes other than proteosomal degradation (Deng et al, 2000
; Pickart and Fushman, 2004
). Structural studies showed that K63 ubiquitin chains are more flexible than K48 chains, providing a biochemical basis for selective recognition of K63-ubiquitinated proteins (Pickart and Fushman, 2004
; Winget and Mayor, 2010
; Fushman and Walker, 2010
). A quantitative proteomics profile of polyubiquitin linkages in yeast showed that all the lysines in ubiquitin can form chains and, with the exception of lysine 63, can directly target proteins to the proteasome for degradation with varying efficiency (Xu et al., 2009b
). While ubiquitin chains of distinct conformations had been defined in yeast, their importance in signaling pathways in metazoan organisms is now being more fully realized. We now highlight some of the recent discoveries that manifest how the diversity of ubiquitin signaling modalities impact immune cell signaling pathways.
The presence of four ubiquitin coding genes and the widespread use of ubiquitin modifications in multiple aspects of cell biology renders strategies for the genetic manipulation of ubiquitin linkages difficult. Chen and colleagues devised a novel ubiquitin replacement strategy for testing the requirements for specific ubiquitin chain linkage in cells. Using an inducible system for the coordinated knockdown of endogenous ubiquitin and expression of mutant ubiquitin in model cell lines, they showed that IL-1 induced NF-kB signaling requires K63 ubiquitin chains. By contrast, TNF induced NF-kB signaling can occur in the absence of K63 (Xu et al, 2009a
). This strategy was also used to show that K63 ubiquitination is required for viral activation of interferon regulatory factor-3 (IRF3) (Zeng et al., 2010
). These studies and others uncovered the physiological importance of K63 ubiquitin chains in immune signaling. In addition, functions of other non-K48 linkages have been described in immune cells.
K33 polyubiquitin chains and TCR signaling
Like K63 polyubiquitin chains, K33 linkages in yeast are relatively resistant to proteasomal degradation, and may thus support nondegradative functions (Xu et al., 2009b
). K33 linkages have recently been described in T cells, where the E3 ubiquitin ligases Cbl-b and Itch appear to cooperatively promote K33-linked ubiquitination of TCRζ. This modification inhibits TCRζ’s phosphorylation and association with the tyrosine kinase Zap-70 and thereby restricts TCR signaling (Huang et al., 2010
). Hence, K33 ubiquitination can disengage TCRζ from TCR signaling without inducing its proteosomal degradation, providing an additional mechanism by which ubiquitination can restrict signals. Utilization of this type of ubiquitin mediated restriction of signals, rather than proteosomal degradation, allows cells to reutilize TCRζ without spending energy on degrading and resynthesizing this protein.
K27 ubiquitination and IKKγ function
K27-linked ubiquitin chains have been described in several settings. The E3 ubiquitin ligase TRAF6, which helps build K63 ubiquitin chains, has been reported to promote K6, K27, and K29 ubiquitination of the Parkinson’s disease proteins DJ-1 and alpha-synucluin, stimulating their accumulation in cytoplasmic aggregates (Zucchelli et al., 2010
). In the setting of host-pathogen interactions, two distinct K27 chain modifications of the NF-kB regulatory subunit IKKγ (also called NEMO) have been described. During viral infections, the attachment of K27 ubiquitin chains to IKKγ by host E3 ubiquitin ligase triparite motif protein 23 (TRIM23) supports IKKγ activation and production of anti-viral IFN-β (Arimoto et al, 2010
). By contrast, during infection with the bacterium Shigella, the attachment of K27 linked ubiquitin chains to IKKγ by bacterial E3 ubiquitin ligase IpaH9.8 promotes its degradation and thus suppression of NF-kB signaling and host defense responses (Ashida et al., 2010
). As bacteria do not possess a ubiquitin conjugation system, Shigella have apparently hijacked the eukaryotic E3 ubiquitin ligase system to suppress host immune defense mechanisms. This exemplifies how pathogens can usurp the ubiquitin system to affect host signaling cascades (Spallek et al., 2009
). Moreover, these two studies identify K27 ubiquitination of two distinct lysines of IKKγ that lead to two different biochemical outcomes.
Linear ubiquitin chains are linked via the N-terminal amino group
In addition to forming ubiquitin chains using one of ubiquitin’s seven lysine residues, so-called “linear” ubiquitin chains can be built via the N-terminal amino group of ubiquitin. Linear chains are assembled by an E3 complex called l
omplex (LUBAC) (Kirisako et al., 2006
), comprised of two E3 ubiquitin ligases, HOIL-1 and HOIP. A ubiquitin associated (UBA) domain in HOIP binds to the ubiquitin-like domain in HOIL-1 to form LUBAC. LUBAC activates the canonical NF-kB pathway by conjugating linear polyubiquitination chains to IKKγ, and HOIL-1 deficient mouse embryonic fibroblasts (MEFs) exhibit reduced TNF- or IL-1β-induced IKK kinase activity (Tokunaga et al., 2009
). Hence, linear polyubiquitin chains appear to be a physiologically important form of ubiquitination. Of note, linear ubiquitin chains were not detected in a quantitative yeast proteomic study, so this aspect of the ubiquitin system may have evolved more recently than yeast (Xu et al., 2009b
). Interestingly, the lysines of IKKγ that putatively undergo ubiquitination with linear ubiquitin chains overlap those identified to undergo K63 ubiquitination. Thus, ubiquitination of individual lysines on substrate proteins might cross-regulate ubiquitination of these same lysines by other chains.
Forked ubiquitin chains can be formed in vitro and in vivo
Most polyubuiqtuin chains are believed to be homogenous, i.e., contain only one type of isopeptide linkage. Recent studies have suggested that a proportion of polyubiquitin chains may be synthesized with mixed linkages, forming forked ubuiquitin chains (Kirkpatrick et al., 2005
; Kirkpatrick et al., 2006
). Forked polyubiquitin chains are resistant to proteasomal degradation and their formation in the cell can be regulated by association with a ubiquitin interacting motif (UIM) protein, suggesting that formation of forked chains may be important in the regulation of normal protein homeostasis, the cellular stress response, and/or in degenerative diseases (Kim et al., 2009
Unanchored ubiquitin chains support NF-kB signals
“Anchored” ubiquitin chains refer to the typical polyubiquitin chains that are covalently attached to substrate proteins. “Unanchored” polyubiquitin chains, by contrast, are free chains that are not attached to any substrate. Unanchored chains can be built by various E3 ubiquitin ligases in vitro. As noted above, TRAF6 works with the E2 UBC13-UEV1A complex to build K63-linked ubiquitin chains. These chains are recognized by the TAK1-TAB1-TAB2 complex, a ubiquitin-dependent kinase complex that phoshorylates mitogen activated protein kinase kinase (MKK) and IκB kinase (IKK)γ. A recent study reported the startling discovery that unanchored K63 ubiquitin chains activate the transforming growth factor associated kinase 1(TAK1) by binding to TAK1 binding protein 2 (TAB2). The presence of unanchored chains in live cells was uncovered by treating immunoprecipitated cell lysates with isopeptidase T, a de-ubiquitinating enzyme that only cleaves unanchored chains (due to its requirement for access to the C-terminal carboxyl group of ubiquitin) (Xia et al, 2009
Another example of the importance of unanchored chains in supporting signal transduction was discovered in the retinoic-acid-inducible gene-1 (RIG-1) pathway. RIG-1-like receptors (RLRs) are involved in viral recognition and trigger signal transduction cascades leading to type I interferon production, and TRIM-25 is an E3 ubiquitin ligase required for the activation of this pathway (Gack et al., 2007
). Sequential binding of viral RNA to RIG-1’s C-terminal regulatory domain and unanchored K63 ubiquitin chain binding to RIG-1’s N-terminal Caspase recruiting domain (CARD) domain may lead to activation of RIG-1 (Zeng et al., 2010
). The unanchored K63 chains that bind to and activate RIG-1 appear to be significantly shorter than the unanchored chains that bind TAK1. These observations raise several questions, including whether unanchored chains are generated as free chains, or whether they are cleaved after being initially built on substrates. If the latter occurs, then additional enzymatic steps must be required. More globally, how and why would unanchored chains, rather than anchored chains, be useful or sufficiently specific in propagating signals?
The answers to these questions should yield surprising insights into the mechanisms by which ubiquitin chains regulate signals.
In summary, a diverse array of physiological polyubiquitin chains provides nearly 10-fold greater biochemical variety than binary phosphorylation events. These varied polyubiquitin chains target modified proteins and signaling complexes for diverse protein-protein interactions such as proteasomal degradation, receptor recycling, signal complex localization, and/or recruitment of downstream signaling proteins. These interactions help support, restrict, or direct signaling cascades toward proper cellular responses. Precise construction and utilization of distinct polyubiquitin chains requires ubiquitin modifying enzymes that can build, edit and degrade these chains as well as ubiquitin sensors that recognize and bind specific chain types.