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Engagement of the T cell receptor (TCR) with its cognate peptide/MHC initiates a cascade of signaling events that results in T cell activation. Limiting the extent and duration of TCR signaling ensures a tightly constrained response, protecting cells from the deleterious impact of chronic activation. In order to limit the duration of activation, T cells must adjust levels of key signaling proteins. This can be accomplished by altering protein synthesis or by changing the rate of protein degradation. Ubiquitination is a process of ‘tagging’ a protein with ubiquitin and is one means of initiating protein degradation. This process is activated when an E3 ubiquitin ligase mediates the transfer of ubiquitin to a target protein. Accordingly, E3 ubiquitin ligases have recently emerged as key regulators of immune cell function. This review will explore how a small group of E3 ubiquitin ligases regulate T cell responses and thus direct adaptive immunity.
Ubiquitination is a post-translational modification that results when ubiquitin is covalently attached to a protein substrate, usually resulting in substrate degradation. Accordingly, the ubiquitination pathway is regulated by several groups of ubiquitin processing enzymes [1–4].
Three groups of ubiquitin processing enzymes have been identified. The E1 activating enzyme processes ubiquitin and transfers it to an E2 conjugating enzyme via a thioester linkage (Fig. 1). The E2 then interacts with an E3 ubiquitin ligase. Among these ubiquitin-processing enzymes, E3 ubiquitin ligases are uniquely responsible for selecting the target proteins and directing the attachment of ubiquitin to the protein substrate. Of the E3 ubiquitin ligases, only members containing the homologous to the E6-AP carboxyl terminus (HECT) domain have intrinsic ubiquitin ligase activity obviating the requirement for an associated E2, while those of the really interesting new gene (RING) domain family must remain associated with an E2 ubiquitin ligase to function. Both HECT- and RING-type E3 ubiquitin ligases induce protein ubiquitination in cells of the immune system and thus regulate immune responses.
T cell activation begins when T cell receptors (TCRs) contact peptide antigen presented by an antigen-presenting cell. For T cells to become fully activated, their co-stimulatory receptors (i.e., CD28) must also be engaged. Together, these events trigger signaling cascades that promote T effector differentiation and cytokine production. In contrast, immunological self-tolerance refers to processes by which immune system cells learn not to attack self-antigens. In T cells, these processes include thymic deletion of cells that respond to self-peptides (negative selection), anergy, and suppression by regulatory T cells.
Several HECT- and RING-type E3 ubiquitin ligases regulate both activation and lymphocyte tolerance. For example both Itch and Cbl-b, an HECT- and RING-type E3 ubiquitin ligase, respectively, are linked to the induction and maintenance of immune self-tolerance [5–15]. While the loss of Cbl-b results in spontaneous autoimmune disease, Itch deficiency leads to abnormal inflammatory responses.
In this review, we will first briefly define the structural domains of Cbl- and neural precursor cell expressed developmentally down regulated 4 (Nedd4)-family members and describe the biological function(s) of these domains. This will be followed by a more detailed account of how these E3 ligases regulate T cell responses. Finally, we will describe how two T cell relevant members of the Nedd4-family, namely Nedd4 and Itch, are regulated.
There are three mammalian members of the Cbl-family of RING-type E3 ubiquitin ligases, c-Cbl, Cbl-b, and Cbl-3. These proteins contain two conserved domains that regulate their E3 ligase activity (Fig. 2); a phosphotyrosine-binding domain (PTB, a.k.a. tyrosine kinase domain) and a RING finger domain (RING) [16, 17]. The PTB domain determines substrate specificity by engaging phosphotyrosine motifs on target proteins. The RING domain binds E2 conjugating enzymes and thus facilitates the transfer of ubiquitin from the E2 to the bound target. Additionally, c-Cbl and Cbl-b have extensive C-terminal extensions containing proline-rich (PR) and ubiquitin-associated (UBA)/Leucine zipper (Leu) domains. These domains bind adaptors and ubiquitin-associated proteins, respectively. UBA domains can also interact with each other, allowing c-Cbl and Cbl-b to form homo- and hetero-dimers . Even so, the functional importance of the UBA domains of Cbl proteins remains controversial [19–21].
Members of the Nedd4-family of HECT-type E3 ubiquitin ligases also share common structural features, including a C2 domain, an HECT domain, and multiple WW domains  (Fig. 3). The amino-terminal C2 domain shares sequence homology with those found in Protein Kinase C (PKC) and Phospholipase C (PLC) proteins . In response to elevated intracellular calcium, the C2 domain of PKC and PLC facilitates the association of these proteins with membranes. While some studies indicate that the C2 domains of Nedd4-family members might have a similar utility , these domains are reported to have other functions. Several studies have shown that C2 domains direct binding of the ligase to adaptor proteins. For example, the C2 domain of Nedd4-2 mediates binding of this HECT-type ligase with the adaptor protein Grb10 . This interaction juxtaposes Nedd4-2 near the insulin-like growth factor receptor (IGF-1R), and promotes IGF-1R ubiquitination . Nedd4-2 also binds Annexin XIIIb via its C2 domain, bringing Nedd4-2 to its well-known target, the epithelial sodium (Na+) channel (ENaC) . Both examples support a role for the C2 domain in directing ligase localization within the cell.
The C2 domain can also directly facilitate an association between ligase and target protein. Smurf1 is a Nedd4-family member that ubiquinates hPEM-2, a guanine nucleotide exchange factor for the Rho GTPase Cdc42 . In order to accomplish this, the C2 domain of Smurf1 binds the pleckstrin homology (PH) domain of hPEM-2. Interestingly, this binding occurs independently of Ca++ mobilization. Taken together, these data support a model in which C2 domains promote E3 ligase function by placing Nedd4-family members in close proximity to their targets.
The C2 domain can also act to inhibit E3 ubiquitin ligase function. In one example, the C2 domain of Smurf2 was shown to form an intramolecular association with its HECT domain . This interaction maintains Smurf2 in an inactive conformation, preventing its ubiquitination of targets as well as its autoubiquitination and subsequent self-destruction. In this case, conformational inhibition preserves both the ligase and its targets.
While auto-inhibition via C2:HECT interactions has been shown to regulate several Nedd4-family members, this typically occurs via the WW domains . Nedd4-family members have 2–4WW domains that can bind motifs within the HECT-domain to promote ligase stability. For example, the WW domains of Nedd4-2 bind to a motif located within its HECT-domain. This type of intramolecular interaction has also been shown for Itch . Thus, conformational auto-inhibition, via binding of the HECT domain to either the C2 or WW domain, could be a general mechanism for controlling the function of this family of E3 ligases.
While Nedd4-family WW domains are capable of repressing enzymatic function and promoting ligase stability, these domains typically determine ligase specificity. WW domains are protein interaction modules found in many eukaryotic proteins. Three types have been classified based on their preference for specific polyproline motifs . The WW domains of HECT-type ligases favor P/LPXY (X denotes any amino acid at this position), but WW domains will also associate with polyproline-rich and PPLP motifs. It should be noted that in some instances WW domains bind proteins that lack these domains [31, 32]. Thus, WW domains, including those of Nedd4-family members, bind motifs that have yet to be identified.
HECT-domain containing E3s have intrinsic catalytic activity and thus can directly ubiquitinate their targets. The HECT domain is the catalytic subunit for ubiquitin transfer, relocating the ubiquitin from its own surface to a lysine residue on its target. E3 ubiquitin ligases can add a single ubiquitin onto one (monoubiquitin) or more target lysines (multimonoubiquitination), or direct the assembly of a chain of linked ubiquitins (polyubiquitination).
The ubiquitin protein has seven lysines (K) that can be used to form polyubiquitination chains; however K63 and K48 chains are most common . Different HECT-E3s employ distinct mechanisms of ubiquitin chain assembly. For example E6AP preferentially forms K48- while Nedd4 typically forms K63-links . Several lines of evidence indicate that proteins tagged with K48-polyubiquitin chains are destined for proteasomal degradation, however the functional importance of K63-linked chains is not clearly defined. While other ubiquitin linkages have recently been described, their importance in T cell function has not been established.
Two members of the Cbl-family of E3 ubiquitin ligases are important regulators of T cell function. Both C-cbl and Cbl-b are expressed in T cells, while Cbl-3 is expressed in epithelial cells of the GI tract and epidermis. This review will focus on how c-Cbl and Cbl-b regulate T cell responses.
C-Cbl is an E3 ubiquitin ligase that controls expression of receptor tyrosine kinases . In T cells, c-Cbl forms a complex with ZAP70 and phosphorylated subunits of the TCR (CD3ζ and CD3δ chains) promoting their ubiquitination [14, 35]. In the thymus, this may act to adjust the threshold for TCR signaling. Interestingly, while c-Cbl deficient thymocytes have enhanced positive selection, negative selection is not markedly impaired. This may be because thymocytes expressing both CD4 and CD8 (double positive) show increased levels of TCR and CD4 proteins on their surface . Paradoxically, once these T cells leave the thymus, they become hyporesponsive, and are less sensitive to TCR-stimulation than wild-type their counterparts. Thus, c-Cbl clearly regulates thymocyte selection.
In a normal T cell response, Cbl-b is recruited to the immunological synapse shortly after TCR activation . Once there, Cbl-b ubiquitinates key transducers of TCR signals, including Vav1 and the p85 subunit of PI3K [38–40] (Fig. 4). Ubiquitination can initiate degradation of the target protein or block its ability to interact with other proteins. Thus Cbl-b dampens T cell activation by reducing the signaling capacity of its target proteins (Fig. 5). Accordingly, mice lacking Cbl-b develop spontaneous autoimmune disease, characterized by autoantibody production and multi-organ lymphocytic infiltration .
In addition to T cell activation, Cbl-b regulates T cell anergy. Cbl-b protein levels increase as a consequence of elevated intracellular calcium (i.e., after ionomycin treatment) and after treatment of cells with calcineurin [41, 42]. Both of these events have been shown to induce anergy. Thus, when these cells are subsequently stimulated with antigen, they are unable to flux calcium or induce other hallmarks of activation. This is because Cbl-b ubiquitinates PLCγ-1 and PKCθ, preventing these important signaling molecules from inducing downstream signaling events such as Ca++ release and NF-κB activation . Consistent with this, Cbl-b−/− T cells are resistant to anergy induction . This may be because, in the absence of Cbl-b, ubiquitination of PLCγ-1 and PKC-θ does not occur. While the precise mechanisms used by Cbl-b are not clear, what is known is that Cbl-b is crucial for the induction of both activation and tolerance.
Because Cbl-b plays such a pivotal role in regulating T cell responsiveness, Cbl-b regulators must also exist. Nedd4 has been established as one such regulator. Its role will be discussed below.
Two members of the Nedd4-family of E3 ubiquitin ligases regulate T cell function, namely Nedd4 and Itch. In vitro data show that both ligases can ubiquitinate, and cause the destruction of, many of the same targets [41, 43, 44]. Thus, until recently these two ligases were thought to function similarly. However, Itch and Nedd4 deficient mice exhibit different phenotypes, suggesting that these two E3 ubiquitin ligases regulate distinct pathways in vivo.
Itch is an important regulator of T cell responses and has been the subject of numerous reviews [5, 45]. Itch was originally discovered because of its proximity to the agouti locus in mice. A mouse strain with a naturally occurring mutation disrupting expression of Itch and the agouti protein (ASP) was identified. In addition to their darkly pigmented coat color, these mice develop an inflammatory condition that leads to constant scratching, thus the mutation was designated as Itchy .
Itchy mutant mice have a T helper 2 (Th2)-mediated disease, characterized by increased interleukin 4 (IL4) production by T cells and increased circulating antibodies, predominantly of the IgG1 and IgE isotypes . JunB is a transcription factor that, among other things, regulates IL4 gene expression [48, 49]. Following T activation, expression of JunB increases for the first 2 h and is then degraded. This degradation is dependent upon Itch ubiquitination of JunB . Thus, in Itchy mutant mice, JunB levels remain high, causing IL-4 to be produced. These results argue that Itch is an important negative regulator of Th2 activation, at least in part, through the ubiquitination and subsequent degradation of JunB (see Fig. 5). The adaptor protein Ndfip1 plays a crucial role in Itch-dependent degradation of JunB. This regulatory mechanism is described in detail below.
Itch also has an important role in regulating NF-κB activity during tumor necrosis factor receptor 1 signaling. In the absence of TNF, NF-κB is maintained in the cytoplasm as an inactive dimer, coupled to the inhibitor of NF-κB (IκB). Following TNF stimulation, receptor interacting protein (RIP) is K63-polyubiquitinated, an event essential for its activation of the IκB kinase complex (IKK) . Once activated, IKK phosphorylates IκB. This releases NF-κB, allowing it to re-locate to the nucleus, where it activates gene transcription.
One of the genes induced by NF-κB is A20, a multifunctional enzyme with deubiquitinating and E3 ligase activity . Once expressed, A20 forms a ternary complex with RIP, Itch, and an adaptor known as TAXBP1 . Together, Itch and A20 edit the K63-polyubiquitin chain on RIP, replacing it with a K48-linked chain. This polyubiquitin chain replacement causes RIP1 degradation and consequent cessation of NF-κB activity. Thus A20 and Itch form a negative feedback loop, terminating NF-kB and limiting transcription of pro-inflammatory genes.
It is not known whether a similar feedback loop regulates NF-kB activation in TCR signaling. In T cells, the IKK activation complex is composed of CARMA1, Bcl10, and MALT1 (a.k.a CBM). It has been shown that CBM components require K63-polyubiquitin linkages to induce IKK activation [54, 55]. A20 was recently shown to interact with one member of this complex, namely MALT1 . Whether Itch and A20 act to inhibit CBM function late in T cell activation is not known.
Itch was recently shown to play an important role in the induction of regulatory T cells (Tregs) . Tregs are a specialized subset of T cells (CD4+CD25+FoxP3+) that suppress activation of conventional CD4+ T cells [58–62]. Accordingly, Treg cells help to maintain tolerance to self-antigens. Naturally occurring Tregs (nTregs) are generated during T cell development in the thymus. Additionally, naïve T cells (CD4+ CD25−) can be induced to convert into Tregs in vitro (iTregs). This conversion involves treating cells with TGFβ, antigen and IL-2. These combined signals trigger expression of the transcription factor FoxP3.
Itchy mice express normal numbers of ‘natural’ Tregs . However, naive T cells from these mice are refractory to TGFβ-mediated Treg induction. This is because the transcription factor transforming growth factor β-inducible early gene-1 (TIEG1) is an Itch target. Importantly, ubiquitination of TIEG1 by Itch does not lead to its degradation. Rather, ubiquitinated TIEG1 and Itch cooperate to induce Foxp3 transcription. Thus, while Itch does not influence nTreg development, Itch regulates TGFβ-mediated conversion into FoxP3 expressing regulatory T cells in vitro. Whether defective Treg conversion contributes to the phenotype of Itch−/− mice has yet to be established.
Several Hect-type E3 ligases have been established as important regulators of T cell activation. Two of these, Itch and Nedd4, share 42% homology and have considerable structural similarity. Both are expressed in peripheral T cells. In vitro, Itch and Nedd4 have been shown to ubiquitinate and degrade similar targets, leading to the speculation that these E3 ligases function similarly. Because Itch is a negative regulator of T cell activation, it has been of interest to determine whether Nedd4 might have a similar role.
Mice lacking Nedd4 are perinatal lethal, due in part, to a growth defect during fetal development . In order to study the role of Nedd4 in immune function, we generated Nedd4−/− fetal liver chimeras (Nedd4−/−FLCh) . Surprisingly, Nedd4−/−FLCh does not mount an effective immune response. In these mice, T cells proliferate poorly in response to antigen and are less likely to produce IL-2, while B cells undergo class switching with lower frequency. From these results, we reasoned that Nedd4 likely promotes T cell activation and enhancement of the adaptive immune response (Fig. 5).
The dissimilarities seen between the immunologic phenotypes of Nedd4−/− and Itch−/− mice suggest that these E3 ligases ubiquitinate unique targets and thus function quite differently in the regulation of T cell responses. It has been shown that the Th2-mediated disease in Itch mutant mice can be explained, in part, by the loss of Itch-mediated degradation of JunB. We have found that the diminished activation of Nedd4−/− T cells can be explained by the ubiquitination and degradation of Cbl-b . These data suggest that Nedd4 is required for T cell activation that triggers adaptive immunity.
Both direct and indirect evidence suggest that Nedd4 and Itch must become activated to be functional . In the cases described thus far, these E3 ligases ubiquitinate their targets only when T cells are activated. While indirect, this suggests that these E3 ubiquitin ligases are regulated by signals transduced by the TCR. While little is known regarding how Nedd4 function is controlled, more is known about Itch. Itch can be regulated by at least four distinct mechanisms, including conformational inhibition, increased expression, phosphorylation, and by association with adaptor proteins.
Signals known to induce anergy also up-regulate Itch protein expression . Importantly, Itch expression is increased after T cell activation . This suggests that Itch may function under both circumstances.
Signals generated by the TCR lead to the phosphorylation and activation of Jun N-terminal kinase (JNK1). Activated JNK1 has been shown to phosphorylate Itch [30, 50]. Itch phosphorylation releases the intramolecular association between the WW and HECT domains, an event that strongly increases the catalytic activity of the E3. The resulting open conformation is believed to promote binding of Itch to an E2 as well as to target proteins in the cell.
Conversely, tyrosine phosphorylation of Itch by the Src-kinase Fyn appears to have an inhibitory effect on Itch function as measured by reduced ubiquitination of JunB . In this example, it is assumed that phosphorylation decreases the binding affinity between Itch and its substrate.
In vitro, Nedd4 and Itch can ubiquitinate targets when incubated in the presence of ATP, E1, E2, and ubiquitin. However, in vivo, HECT type E3 ligases may rely on adaptor proteins to function effectively. Using yeast-2-hybrid screens or similar methods, several laboratories have identified adaptors that bind members of the Nedd4-family. Several of these studies used the WW domains of either Nedd4 or Nedd4-2 [68, 69].
Nedd4-family interacting protein 1 (Ndfip1, N4WBP5) was recently identified by one of these screens because of its ability to bind the WW domains of Nedd4 . Ndfip1 contains two PPXY motifs that facilitate this interaction. When over-expressed in COS cells, Ndfip1 was also shown to interact with the WW domains of other HECT-type family members, including WWP2 and Itch.
We recently described the phenotype of mice lacking Ndfip1 . By 6 weeks of age, Ndfip1−/− mice develop a severe Th2-mediated inflammatory disease of the skin and lung . This phenotype is due, in part, to a defect in T cells that lead to production and secretion of IL-4, a Th2 cytokine that promotes atopic disease. IL-4 production by Th2 effectors could account for the high levels of IgE in the serum of Ndfip1−/− mice. It could also explain the presence of eosinophils in the inflammatory lesions.
The phenotype of Ndfip1−/− mice closely resembles that of Itchy mice, indicating that Ndfip1 might regulate Itch. Supporting this, we found that in wild type T cells, Ndfip1 binds Itch and promotes Itch function. Association between Ndfip1 and Itch facilitates the ubiquitination and degradation of a well-described Itch target, JunB. This prevents JunB from driving Th2 cytokine production. This explains why, in the absence of Ndfip1, Itch is expressed but JunB is not degraded. In T cells lacking Ndfip1, high levels of JunB likely induce IL-4 production and Th2-mediated disease.
Ndfip1 likely regulates Nedd4-family members in addition to Itch. Ndfip1−/− mice have a much more severe phenotype than their Itchy counterparts. Ndfip1−/− mice begin to show evidence of skin inflammation at 6 weeks of age and do not survive beyond 14 weeks of age. Itch deficient mice, on the other hand, do not develop symptoms until they are 8 month old. This suggests that Ndfip1 regulates another Nedd-4 family member, likely one that inhibits T cell activation. There are nine Nedd4-family members expressed in mammalian cells. These include Nedd4, Nedd4-2, Itch, WWP1, WWP2, SMURF1, SMURF2, and NEDL1 and NEDL2. While Ndfip1 can bind to several of these ligases in vitro, such interactions have not been confirmed in vivo . Whether Nedd4-family members other than Nedd4 and Itch are expressed and function in T cells is not known.
Based on their ability to bind one or more members of the Nedd4-family and because they have a similar domain structure to Ndfip proteins, several families of potential adaptor proteins have been described. Ndfip1 has one closely related family member, Ndfip2 . Like Ndfip1, Ndfip2 expression increases in activated T cells. Whether Ndfip2 regulates E3 ligase function in T cells is not yet known.
While members of the Ndfip-family appear to positively regulate ubiquitin ligase function, another adaptor, N4BP1, was shown to inhibit Itch function . N4BP1 is similar to Ndfip1 in that it contains multiple WW domain-binding motifs, these likely form docking sites for Nedd4 and Itch. Unlike Ndfip1, N4BP1 has no obvious transmembrane regions, suggesting that it resides in the cytosol. This raises the possibility that N4BP1 represents a soluble adaptor that could sequester Itch within the cytosol. It is not known whether N4BP1 is expressed in T cells. Furthermore, inhibition of Itch by N4BP1 has yet to be established in an in vivo setting.
Although Itch and Nedd4 are structurally similar, several lines of evidence suggest that Nedd4 is regulated quite differently from Itch. For example, while Itch is upregulated by T cell stimulation, Nedd4 protein expression is evident in resting T cells and its levels do not rise after TCR triggering (our unpublished results). Furthermore, while Itch is phosphorylated on serine and threonine residues found in a region near its N-terminus, a similar region does not exist within Nedd4, suggesting that Nedd4 is phosphorylated differently from Itch.
Like Itch, Nedd4 may be regulated by adaptors. Several families of adaptors have been described that bind Nedd4 in vitro. One of these, GRB10, associates with the C2 domain of Nedd4, and has been shown to have substrate-dependent positive or negative influence on Nedd4 function [25, 26, 74].
The immune response is a powerful protective process used by organisms to combat pathogen invasion. Without it, the animal would quickly succumb to disease. Conversely, too strong of a response leads to autoimmune or inflammatory disease. T cells monitor the immune response in three important ways: they direct the response toward the pathogen and away from self, they determine the duration of the response, and they retain memory of the pathogen to prevent subsequent invasion. Thus, it is important that we understand how T cells are regulated.
Ubiquitination is an indispensable component of processes that direct T cell function. Accordingly, E3 ubiquitin ligases are key players in T cell activation and the consequent development of an adaptive immune response. Additionally, E3 ligases regulate immunologic tolerance to ensure that T cells remain quiescent toward self. To date, E3 ubiquitin ligases involved in T cell regulation include several members of RING-type ligase family (c-Cbl, Cbl-b, GRAIL) as well as members of the HECT-type ligases (Nedd4 and Itch). Not surprisingly, proteins that regulate these ligases, i.e., adaptor proteins and kinases, also influence T cell responses.
In many cases, the mechanistic details of these ubiquitination pathways remain obscure. While certain players have been identified for a given pathway, many others have not. Defining key players in E3 ubiquitin ligase pathways will provide novel therapeutic targets that could be used to treat patients with autoimmune or inflammatory diseases. Thus, the E3 ubiquitin ligases and their regulators will continue to be studied to better understand how these proteins work.
Denise L. Gay, The Children’s Hospital of Philadelphia, Joseph Stokes, Jr. Research Institute, 3615 Civic Center Blvd, Philadelphia, PA 19104, USA.
Hilda Ramón, The Children’s Hospital of Philadelphia, Joseph Stokes, Jr. Research Institute, 3615 Civic Center Blvd, Philadelphia, PA 19104, USA. University of Pennsylvania, Philadelphia, PA, USA.
Paula M. Oliver, The Children’s Hospital of Philadelphia, Joseph Stokes, Jr. Research Institute, 3615 Civic Center Blvd, Philadelphia, PA 19104, USA. University of Pennsylvania, Philadelphia, PA, USA, Email: paulao/at/mail.med.upenn.edu.