A Family of Signal-Responsive E3 Ubiquitin Ligases Mediating TLR Signaling and T-Cell Tolerance

REVIEW

Peli: a family of signal-responsive E3 ubiquitin ligases mediating TLR signaling and T-cell tolerance

Wei Jin1, Mikyoung Chang2 and Shao-Cong Sun2

E3 ubiquitin ligases play a crucial role in regulating immune receptor signaling and modulating immune homeostasis and activation. One emerging family of such E3s is the Pelle-interacting (Peli) proteins, characterized by the presence of a cryptic forkhead-associated domain involved in substrate binding and an atypical RING domain mediating formation of both lysine (K) 63- and K48-linked polyubiquitin chains. A well-recognized function of Peli family members is participation in the signal transduction mediated by Toll-like receptors (TLRs) and IL-1 receptor. Recent gene targeting studies have provided important insights into the in vivo functions of Peli1 in the regulation of TLR signaling and inflammation. These studies have also extended the biological functions of Peli1 to the regulation of T-cell tolerance. Consistent with its immunoregulatory functions, Peli1 responds to different immune stimuli for its gene expression and catalytic activation. In this review, we discuss the recent progress, as well as the historical perspectives in the regulation and biological functions of Peli. Cellular & Molecular Immunology (2012) 9, 113–122; doi:10.1038/cmi.2011.60; published online 6 February 2012

Keywords: Peli; Pellino; ubiquitination; NF-kB; TLR; T-cell tolerance

INTRODUCTION ubiquitin-specific proteases, termed deubiquitinases.3 The specificity of Ubiquitination is a post-translational mechanism of protein modifica- ubiquitination is determined by E3s, a large family of proteins that tion that has long been recognized as a mechanism mediating protein interact with both specific substrates and E2s and function to transfer degradation in the 26S proteasome.1 This hot research field has become ubiquitin from E2s to the substrates.1 Based on their functional even hotter during recent years due to the involvement of ubiquitina- domains, E3s are divided into two major classes: the RING (really tion in various non-degradative molecular events, including protein– interesting new gene) finger E3s and HECT (homologous to E6-AP protein interactions, intracellular protein trafficking, DNA repair and C-terminus) domain E3s, although atypical E3s have also been iden- activation of signaling molecules.2 Ubiquitination involves the covalent tified.4 TheRINGfingerE3sarebyfarthelargestandalsomostcomplex attachment of the small ubiquitin protein to the lysine residues of family of ubiquitin ligases, with about 579 members being annotated in substrate proteins, as either monomers (monoubiquitination) or the human genome.5 Consistently, the RING E3s have been linked to a polymers (polyubiquitination).1 Formation of polyubiquitin chains large variety of physiological and pathological processes.6 Among the involves the linkage of the C-terminus of one ubiquitin to an internal newly characterized RING E3s are the Pelle-interacting (Peli, also called lysine residue of another ubiquitin. Since each ubiquitin has seven Pellino) proteins, a family of evolutionally conserved immune regulators lysines, all of which can be used for the chain linkage, there are a large thought to regulate signaling from Toll-like receptors (TLRs) and IL-1 7,8 variety of polyubiquitin chains. In addition, ubiquitins can be con- receptor (IL-1R). Recent gene targeting studies have provided new 9,10 nected through head-to-tail ligation, yielding linear ubiquitin chains. insights into the in vivo biological functions of Peli. Important pro- The different ubiquitin chains may mediate distinct biological func- gress has also been made to elucidate the biochemical mechanism medi- 11–13 tions. In particular, lysine 48 (K48)-linked polyubiquitin chains pri- ating signal-induced Peli activation. Inthisreview,wediscussthe marily target proteins for proteasomal degradation, whereas K63-linked recent progress on the regulation and biological functions of Peli in the polyubiquitin chains mediate non-degradative functions, including context of immune receptor signaling and inflammation. assembly of signaling complexes and activation of protein kinases.2 The ubiquitination reaction is catalyzed by the sequential action of E3S IN THE REGULATION OF TLR SIGNALING AND NF-kB three enzymes: the ubiquitin-activating enzyme (E1), the ubiquitin-con- ACTIVATION jugating enzymes (E2s) and the ubiquitin ligases (E3s).1 This reaction is An important function of K63 type of ubiquitination is mediating reversible, since the ubiquitin chains can be deconjugated by a family of activation of the NF-kB signaling pathway.14,15 NF-kB represents a

1Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA and 2Department of Immunology, The University of Texas MD Anderson Cancer Center, New York, NY 10065, USA Correspondence: Dr W Jin, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. E-mail: [email protected] or Dr S-C Sun, The University of Texas MD Anderson Cancer Center, 7455 Fannin Street, Box 902, Houston, TX 77030, USA. E-mail: [email protected] Received 1 December 2011; revised 26 December 2011; accepted 28 December 2011 Peli: immunoregulatory E3 ubiquitin ligases W Jin et al 114

family of transcription factors that participates in the regulation of pathway, TRIF stimulates activation of the IKK-related kinases, TBK1 multiple biological processes, including immune response and inflam- and IKKe, and their downstream transcription factor IRF-3, leading to mation, cell growth and survival, and oncogenesis.16–18 NF-kBis induction of the type I interferons (IFNs).25 This antiviral pathway normally sequestered in the cytoplasm via physical association with also requires K63 ubiquitination, which is mediated by the E3 ubiqui- specific inhibitory proteins, IkBs. Activation of NF-kB typically tin ligase TRAF3.27,28 involves degradation of a prototypical IkB member, IkBa, and rapid In addition to TRAF6 and TRAF3, several other E3 ubiquitin nuclear translocation of NF-kB, although a non-canonical pathway is ligases have been implicated in the positive or negative regulation also involved for NF-kB activation in specific biological processes.19 of TLR/IL-1R signaling.29–32 In particular, a recent study suggests a The IkBa degradation is triggered through its phosphorylation by a role for the cIAP1 and cIAP2 in the regulation of MyD88-depend- multisubunit IkB kinase (IKK) composed of two catalytic subunits ent TLR signaling.31 cIAPs are RING E3s with both K63 and K48 (IKKa and IKKb) and a regulatory subunit, NEMO (NF-kB essential types of ubiquitin ligase activity. In response to lipopolysaccharide modulator).16,17 IKK mediates NF-kB activation by integrating signals (LPS)-mediated TLR4 stimulation, cIAPs are recruited to the from a large variety of innate immune receptors as well as the T- and B- MyD88 signaling complex, where they mediate K48 ubiquitination cell antigen receptors. The activation of IKK by most immune recep- and degradation of TRAF3. Although TRAF3 is required for type I tors requires transforming growth factor-b (TGF-b)-activated kinase IFN signaling,27,28 it serves as a negative regulator in the MyD88- 33 1 (Tak1), a ubiquitin-dependent kinase that activates both IKK and dependent TLR pathway. TRAF3 degradation is essential for the MAP kinases (MAPKs).20–22 Like IKK, Tak1 exists as a complex con- dissociation of the MyD88 signaling complex from the receptor, a taining two adaptor proteins: Tab1 and Tab2 (or its homologue Tab3). step that is required for stimulating the TRAF6-dependent activa- 31 A common feature of Tab2 (or Tab3) and NEMO is the presence of a tion of Tak1 and MAPKs. The recent demonstration of Peli pro- ubiquitin-binding domain that specifically binds to K63-linked poly- teins as RING E3s adds another family of E3s that regulates TLR ubiquitin chains.14,15,23 Such ubiquitin-binding activity is crucial for signaling and inflammation. the recruitment of Tak1 and IKK to upstream signaling adaptors that are conjugated with K63-linked polyubiquitin chains. Within the sig- PELI PROTEINS AS EVOLUTIONALLY CONSERVED IMMUNE naling complexes, both Tak1 and the IKK regulatory subunit NEMO REGULATORS 14,24 are also conjugated with K63-linked ubiquitin chains. It is gene- Discovery of Peli as a Drosophila immune regulator rally believed that the binding of and/or conjugation with ubiquitin Lower animals like insects do not have adaptive immune system, but chains may trigger conformational changes in the kinases and, they have efficient innate immunity to deal with microbial infec- thereby, lead to their catalytic activation. tions.34,35 Early studies by Hans Boman and colleagues demonstrated The ubiquitin-dependent NF-kB activation has been extensively that in response to bacterial infection, the silkmoth Hyalophora cecro- studied in the TLR/IL-1R signaling pathways. TLRs form one of the pia rapidly produces a set of antimicrobial peptides that form an major families of pattern-recognition receptors, which also include important part of its humoral immunity.36 A connection between the RIG-I-like receptors, the NOD-like receptors and the C-type lectin the insect antibacterial response and mammalian innate immunity 25 receptors. By recognizing specific pathogen-associated molecular was first indicated by the finding that H. cecropia has an NF-kB-like patterns, the pattern-recognition receptors mediate induction of transcription factor, cecropia immune factor (CIF), which responds to innate immunity to form the first line of host defense against infec- bacteria and appears to mediate the induction of the antibacterial tions. Both human and mice have multiple TLR members (10 for genes.37,38 This idea was later on confirmed by the identification of human and 12 for mice), which detect and respond to a large array an NF-kB analogue, Dif (dorsal-related immunity factor) from of pathogen-associated molecular patterns, ranging from surface Drosophila melanogaster.39 Dif, together with Dorsal and Relish, form 25 molecules to nucleic acids of the invading microbes. In response the NF-kB family in Drosophila that mediates the induction of various to ligand stimulation, TLRs signal through intracellular adaptors, antimicrobial genes.34 including the common adaptors MyD88 and TRIF. While TRIF medi- Activation of Drosophila NF-kB is mediated by pattern recognition ates signaling from TLR3 and TLR4, MyD88 transduces signals from receptors, Toll and immune deficiency, which respond to fungi and all TLRs, except TLR3, as well as from the IL-1R. A hallmark of the bacteria, respectively.34,35 The Toll pathway of Drosophila is analogous MyD88-dependent TLR/IL-1R pathway is the involvement of evolu- to the mammalian IL-1R/TLR pathway, whereas the immune defi- tionarily conserved IL-1R-associated kinases (IRAKs), including ciency pathway resembles the mammalian tumor-necrosis factor IRAK1, IRAK2, IRAK3 (also named IRAK-M) and IRAK4.25 In res- receptor (TNFR) pathway. In particular, the signal transduction of ponse to ligand stimulation, IRAK family members are rapidly Toll involves recruitment of several intracellular components, includ- recruited to the IL-1R/TLR, via the adaptor MyD88, and mediate ing Drosophila MyD88, Tube and Pelle. Pelle is a homologue of mam- activation of a downstream factor TRAF6. TRAF6 functions as both malian IRAK1, characterized by a kinase domain and an N-terminal an adaptor and a K63-specific E3 ubiquitin ligase and amplifies the IL- death domain. Although Tube has no obvious counterpart in the 1/TLR signal by stimulating several downstream signaling pathways, mammalian system, a recent study suggests that Tube originates from including those leading to activation of NF-kB and three families of an ancestral gene related to IRAK4.40 Consistently, Tube functionally MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal resembles IRAK4 to interact with and activates Pelle.41 Thus, the kinase (JNK) and p38. These signaling pathways act cooperatively Drosophila Tube/Pelle pair may be considered the functional equiva- in the induction of genes encoding proinflammatory cytokines lent of mammalian IRAK4/IRAK1. and chemokines. The TRIF-dependent TLR pathway also targets the Drosophila Peli (Pellino, hereafter named dPeli) was identified as a activation of NF-kB, and this is dependent on an adaptor, receptor- Pelle-interacting protein in yeast two-hybrid screening aiming to interacting protein 1 (RIP1).26 TRIF stimulates the conjugation of identify downstream signaling molecules connecting Pelle to the activa- K63-linked polyubiquitin chains to RIP1, which in turn is important tion of NF-kB.42 dPeli is a protein of 424 amino acids that shares about for recruitment and activation of IKK.26 In addition to the NF-kB 50% identity and 70% homology with its counterpart in Caenorhabditis

Cellular & Molecular Immunology Peli: immunoregulatory E3 ubiquitin ligases W Jin et al 115 elegans, suggesting Peli to be an evolutionarily conserved gene. JNK and p38) and the transcription factors AP-1 and CREB, although Interestingly, the dPeli–Pelle interaction requires the kinase activity they do not activate NF-kB.51,52 On the other hand, another study of Pelle, and Peli may preferentially bind to autophosphorylated demonstrates that the shorter isoform of Peli3 (Peli3b) negatively Pelle.42 The function of dPeli in Drosophila innate immunity has been regulates IL-1-stimulated activation of NF-kB and JNK.53 This latter studied recently using dPeli mutant flies (Pli).43 Pli flies have impaired study suggests that Peli3b mediates K63 ubiquitination of IRAK1 at induction of the antibacterial peptide Drosomycin and reduced sur- K134. Since this site is also used for conjugating K48-linked ubiquitin vival in response to infection by the gram-positive bacterium chains, the Peli3b-mediated K63 ubiquitination of IRAK1 inhibits the Micrococcus luteus. Consistently, overexpression of dPeli induces the K48 ubiquitination and degradation of IRAK1 upon IL-1 stimulation. expression of Drosomycin. Overall, these findings suggest that dPeli is a The degradation of IRAK1 seems to be required for IL-1-stimulated positive regulator of Drosophila innate immunity against certain strains activation of Tak1 and its downstream signaling events, since a pro- of gram-positive bacteria. teasome inhibitor (MG132) inhibits IRAK1 degradation and also blocks activation of IKK.53 This model is intriguing, although more Mammalian Peli members studies are required for further validation using different cell systems, The mammalian Peli members were initially identified based on data- particularly primary cells derived from Peli3 knockout (KO) mice. In base search,44 and subsequent cloning revealed two highly homolo- terms of the discrepancies between the different studies, it is possible gous mouse Peli members (Peli1 and Peli2).45 The function of Peli was that at least some of the differences were attributed to the use of first reported in a study with the mouse Peli2. Peli2 is abundantly different experimental systems. expressed in non-lymphoid organs but only weakly expressed in spleen and thymus. Analogous to dPeli, Peli2 interacts with IRAK-1, PELI PROTEINS ARE E3S WITH A NEW RING AND A CRYPTIC which occurs in an IL-1 inducible manner. In a mouse embryonic FORKHEAD-ASSOCIATED (FHA) DOMAIN fibroblast cell line (C3H10T1/2) and a human embryonic kidney cell E3 function of Peli line (HEK293), reduction of Peli2 expression by antisense RNA inhi- Peli proteins were initially thought to serve as scaffold proteins that bits induction of the chemokine gene IL-8 by IL-1 and the TLR4 ligand regulate the dynamic assembly of intermediate signaling complexes in 46 LPS in a NF-kB-dependent manner. The more detailed mechanism the IL-1R pathway.47 Although how Peli exerts such functions remains of Peli function was revealed in another study using HEK 293 cells incompletely understood, the discovery of Peli proteins as E3 ubiqui- 47 stably expressing the IL-1R. In response to IL-1 stimulation, Human tin ligases provides critical insights into the biochemical mechanism of Peli1 interacts with both IRAK1 and IRAK4 upon IL-1 stimulation. It Peli action.54 Initial sequence analyses reveal that the C-terminal por- appears that Peli1 functions as a scaffold protein involved in the tion of Peli members contain two conserved Cys-Gly-His motifs and formation of an intermediate IL-1R signaling complex composed of two conserved Cys-Pro-X-Cys motifs, reminiscent of C3HC4 RING- 47 IRAK4, IRAK1 and TRAF6. It has been proposed that Peli1 may like zinc finger domains.44,47 By sequence alignments with the C3HC4 47 induce the release of the IRAK complex from the receptor, a step RING consensus sequence,55 Beyaert and colleagues demonstrate that required for the activation of downstream signaling events by IL-1 and although Peli proteins do not contain a typical C3HC4 RING, they 48 TLRs. RNAi-mediated Peli1 knockdown suggests that this Peli possess a closely related motif termed CHC2CHC2 RING.54 member is required for IL-1-stimulated activation of NF-kB and The E3 function of Peli protein was first demonstrated by the find- induction of IL-8. It is currently unclear whether Peli1 indeed is ing that all Peli members were able to induce the polyubiquitination of required for mediating the release of IRAK complex from IL-1R or IRAK1 when overexpressed in mammalian cells.54 This function of TLRs and how Peli1 could possibly regulate this important step of IL- Peli proteins is dependent on the presence of an intact RING-like 1R/TLR signaling. structure. Since the RING domain is dispensable for Peli interaction Despite the uncertainties regarding the precise mechanism of with IRAK1, these findings strongly suggest that Peli members func- Peli function, it is clear that interaction with IRAK1/4 and IRAK- tion as RING E3s that mediate IRAK1 ubiquitination.54 The E3 func- associated factors is a common property of the different Peli members. tion of Peli proteins was later on confirmed by using a cell-free Like the human Peli1 and mouse Peli2, human Peli2 binds to IRAK1 ubiquitination system.56,57 These later studies also reveal that Peli and IRAK4,49 as well as to TRAF6 and Tak1.50 Similarly, the two iso- can function together with the Ubc13-Uev1a dimeric E2 ubiquitin- forms of Peli3, Peli3a and Peli3b, interact with IRAK1, TRAF6, Tak1, conjugating enzyme to catalyze K63-linked polyubiquitin chains.56,57 as well as to the NF-kB-inducing kinase.51,52 It is important to note However, Peli does not seem to be an exclusively K63-specific E3 though that these binding assays were performed in transfected mam- ubiquitin ligase, since it also catalyzes the production of K48 and malian cells. Since the different Peli-interacting proteins are often K11 types of polyubiquitin chains when working with other E2s, such present in the same complexes, it is likely that some of these proteins as UbcH3, UbcH4, UbcH5a and Ubc5b.57 The ability of Peli to utilize may associate with Peli indirectly via other proteins, like IRAK1. different E2s suggests a high level of versatility in Peli function. As will In contrast to the consistent findings on the Peli-interacting factors, be discussed in a later section, both the K63- and K48-specific E3 the functional studies of Peli members have generated some discre- functions of Peli1 have been linked to important physiological func- pancies. For example, whereas one study suggests that Peli2 activates tions.9,10 Which E2s are required for in vivo functions of Peli and how NF-kB,46 another study suggests that Peli2, but not Peli1, mediates the chain specificity of Peli-mediated ubiquitination is regulated activation of the MAPK JNK and induction of its downstream tran- remains an intriguing topic of investigation. scription factors AP-1 and Elk-1.50 Yet, another study suggests that Although Peli proteins clearly have E3 ligase activity, their mode of Peli2 is not required for IL-1-induced NF-kB target gene expression action is not well understood. It has been shown that the K63-linked and that overexpressed Peli2 does not reproducibly activate MAPK polyubiquitin chains catalyzed by Peli1 and Ubc13-Uev1a are not nor induce reporter gene expression through specific transcription attached to IRAK1 or Peli1 in vitro, but rather stay as free forms.11,57 factors.49 The biological functions of Peli3 seem to be also complex. This is in sharp contrast to the efficient conjugation of K63 polyubi- Some studies reveal that both isoforms of Peli3 activate MAPKs (ERK, quitin chains to IRAK1, when it is coexpressed with Peli in cells.54,56,57

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Since Peli can mediate ubiquitination together with different E2s, it is activity of Peli proteins is also regulated by phosphorylation.11–13,57 It thought that Peli-mediated IRAK1 ubiquitination may be a two-step was noted initially that IRAK1 and IRAK4 phosphorylate Peli mem- process involving two different E2s. Peli1 may first act with a different bers in transfected cells.49,54 Using recombinant proteins and in vitro E2 to mediate IRAK1 monoubiquitination and then pair with Ubc13- ubiquitination assays, Cohen and colleagues demonstrate that the Uev1a to extend the ubiquitin chains with K63 linkage.11 Although phosphorylation of Peli proteins greatly stimulates their E3 ligase this idea needs to be tested by additional experiments, the poor in vitro activity.11,57 How phosphorylation activates Peli remains unclear, ubiquitin-conjugating activity of the Peli1/Ubc13/Uev1a system has but it appears that this mechanism regulates the general E3 function, also been observed with another substrate, RIP1, which is efficiently instead of specific E2 usage, since Peli1 phosphorylation stimulates its ubiquitinated by Peli1 in vivo.9 Despite these uncertainties, the accu- E3 function regardless of which E2 is used.11 mulating studies have more and more firmly confirmed the E3 ubi- The functional study of Peli1 phosphorylation is complicated by the quitin ligase function of Peli proteins. The functional significance of presence of multiple potential phosphorylation sites.11 At least in vitro, the RING-like domain of Peli is also emphasized by a recent finding IRAK1 and IRAK4 phosphorylate Peli1 at up to 10 serines or threo- that this structural domain is evolutionally conserved from human to nines, the simultaneous mutation of all of which is required to prevent 58 sponge Peli proteins. Peli1 activation.11 A caveat of such mutagenesis approach is the possible cause of conformational changes that could have affected the Substrate binding by an FHA domain IRAK binding or E3 ubiquitin ligase activity of Peli1. Nevertheless, In addition to their E2-binding domain (RING or HECT), single- this in vitro study provides intriguing insights that may help predict subunit E3s also contain a domain that mediates substrate binding. the mechanism of Peli regulation. Most interestingly, the major phos- It is known that the C-terminal RING domain of Peli is not involved in phorylation sites of Peli1 are clustered within two regions: the first the Peli–IRAK1 interaction.54 Initial sequence analyses did not reveal region (amino acides 76–86) correlates with the wing-like appendage any notable structural domains within the N-terminal region of Peli of the FHA domain and the second (containing threonine-288 and that binds IRAK1. However, a structural study based on X-ray crys- serine-293) is located just before the RING domain. Since FHA binds tallography led to the discovery of a cryptic FHA domain located in the to phosphothreonine-containing motifs, it would be interesting to test N-terminal region (amino acids 15–275) of Peli2.59 A unique feature whether phoshorylation of Peli1 promotes its dimerization or intra- of FHA domains is their interaction with protein motifs containing a molecular interaction, which in turn could serve as a mechanism of phosphorylated threonine residue.60 Compared with the typical FHA Peli activation. It is also possible that phosphorylation of the FHA domains, the Peli2 FHA domain contains two insertion regions that region of Peli may promote its interaction with IRAK1 or other sub- form a wing-like appendage. Nevertheless, the cryptic FHA domain of strates/regulators. Peli2 has the conserved b-strands and all of the key amino-acid resi- Recent studies reveal that in addition to IL-1, many other immune dues known to be involved in phosphothreonine binding.59 Indeed, stimuli, such as ligands of TRIF-dependent and MyD88-dependent the FHA domain of Peli2 binds to phosphothreonine-containing pep- TLRs and tumor-necrosis factor-a (TNF-a), stimulate the phosphor- tides, as well as to the IRAK1 protein expressed in HEK293 cells, and ylation and activation of Peli112,13 (Figure 1). Interestingly, despite the these interactions rely on the conserved ligand-binding amino-acid implication of IRAK1 as the major kinase of Peli, IRAK1 is required 59 residues. Interestingly, an earlier biochemical study identified a only for Peli1 activation by IL-1, whereas the IKK-related kinases, motif of Peli3 (amino acids 43–47; KYGEL) that is critical for binding TBK1 and IKKe, mediate the phosphorylation and activation of 52 of Peli3 to IRAK1. This motif, which is equivalent to amino acids 18– Peli1 in the TNFR and TLR pathways. Traditionally thought to be 59 22 of Peli2, happens to form the first b-strand of the FHA core. This TRIF-dependent kinases, TBK1 and IKKe have now been shown to finding further suggests that FHA is a functional domain in all Peli also respond to signals from the MyD88-dependent TLRs, as well as members. the TNFR.12,13 Since both IL-1R- and MyD88-dependent TLRs activ- The discovery that Peli contains a phosphothreonine-binding ate IRAK1, it is surprising that IRAK1 is important only for the IL-1R domain provides molecular basis to the previous findings that Peli– pathway. Whether this is a cell type-specific function or involves a yet- 46,47,51 IRAK1 interaction is induced by IL-1R/TLR signals and requires to-be-defined mechanism remains to be investigated. Since Peli1 is 42,49,54 the kinase activity and phosphorylation of IRAK1. Collectively, also activated in T cells,10 it raises the question of which kinase(s) the results of this structural study, along with those of the previous mediates Peli1 phosphorylation and activation by the T-cell receptor biochemical analyses, suggest an intriguing model for the inducible (TCR) and the T-cell costimulatory molecule CD28 (Figure 1). interaction between Peli and IRAK1. In response to IL-1R/TLR signals, IRAK1 becomes phosphorylated by IRAK4, and phosphorylated Ubiquitination and sumoylation IRAK1 recruits Peli via FHA–phosphothreonine interaction. The When co-expressed with IRAK1 in mammalian cells, Peli not only recruited Peli then catalyzes K63 ubiquitination of IRAK1, thereby induces ubiquitination of IRAK1, but also becomes ubiquitinated allowing the recruitment and activation of downstream kinases Tak1 52 itself. Since IRAK1 activates the E3 ligase of Peli, it is likely that and IKK. Since Peli also interacts with various other proteins, future the Peli ubiquitination represents auto-ubiquitination, although the studies should examine whether the same or different mechanisms involvement of other E3 ligases is also possible. At least in vitro, mediate Peli interaction with other substrates or regulatory proteins. IRAK1-activated Peli1 undergoes auto-ubiquitination in the presence of the E2 UbcH4.11,57 Since Peli can conjugate K48- and K11-linked MECHANISMS OF PELI REGULATION polyubiquitin chains, known to mediate protein degradation, the Activation by phosphorylation auto-ubiquitination of Peli may contribute to its inducible degrada- How the function of E3s is regulated is still poorly understood. tion as shown for Peli3.7,52 The Peli1 auto-ubiquitination occurs at Nevertheless, evidence is emerging that phosphorylation plays an three major K residues, including K169, K202 and K266. K169 and important role in regulating the E3 activity and ligand-binding func- K202 are located in the wing-like appendage and the FHA core, tion of E3s.61–64 Several recent studies demonstrate that the E3 ligase respectively, whereas K266 is located C-terminal to the FHA

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kinases, TBK1 and IKKe, which in turn induce the nuclear translocation of the transcription factor IFN-regulatory factor 3 (IRF3) and induction of type I IFNs.68,69 Peli1 induction requires TBK1/IKKe and the downstream transcription factor IRF3, but it does not involve the type I IFNs.12 It is thus likely that Peli1 is a target gene of IRF3. Since Peli1 is also induced in T cells by the TCR and CD28 costimulatory signals,10 it raises the question of which signaling pathway and transcription factor mediates the T cell-specific Peli1 induction. The induction of Peli1 in T cells appears to serve as an autoregulatory mechanism to prevent aberrant NF-kB activation and T-cell activation. As will be discussed later, Peli1 negatively regulates T-cell activation by targeting the ubiquitination and degradation of a major NF-kB family member, c-Rel.10 However, since Peli1 is a positive regulator in innate immune cells, it is currently unclear how the inducible expression of Peli1 contributes to the regulation of TLR signaling. The expression level of Peli may also be subject to regulation at post- transcriptional levels, as suggested by the recent identification of Peli1 as a target of a microRNA, miR-21.70 The level of Peli1 mRNA is inversely correlated with that of miR-21 during liver regeneration. Sequence analyses using the TargetScan program revealed a putative target sequence of miR-21 in the 39-untranslated region of Peli1. Insertion of the 39-untranslated region of Peli1 downstream of a luci- Figure 1 Signal-induced Peli1 activation. Peli1 exists as an inactive form but ferase reporter gene renders the reporter sensitive to miR-21-mediated becomes activated in response to signals from different immune receptors. Peli1 inhibition. Overexpression of miR-21 in HEK293 cells also inhibits the activation involves its phosphorylation by IRAK1 and the IKK-related kinases induction of the NF-kB reporter gene.70 Although this finding is TBK1 and IKKe. Whereas IRAK1 mediates Peli1 activation by IL-1R, TBK1 and IKKe mediate Peli1 activation by TLRs and TNFR. It remains unclear how Peli1 is potentially interesting, it is yet to be examined whether the level of activated in T cells by the TCR and CD28 signals. IKK, IkB kinase; IRAK, IL-1R- endogenous Peli1 mRNA and protein is indeed regulated by miR-21 associated kinase; Peli1, Pelle-interacting protein 1; TCR, T-cell receptor; TLR, and whether miR-21-mediated NF-kB inhibition is mediated through Toll-like receptor; TNFR, tumor-necrosis factor receptor. Peli1 down regulation.

11 IN VIVO FUNCTIONS OF PELI1 REVEALED BY GENE domain. It would be interesting to examine whether Peli1 auto- TARGETING STUDIES ubiquitination interferes with its binding to IRAK1 or other substrates. If this is indeed the case, it will suggest that Peli1 ubiquitination Gene targeting in mice is a powerful approach to study the physiological functions of genes. However, one technical issue of this may serve as a mechanism to both terminate its function and target its approach in studying the function of gene families is the functional proteolysis. redundancy among family members. The extensive sequence/struc- A recent study reveals that Peli1 is also conjugated with the small tural homology between the Peli family members predicts functional ubiquitin-related modifier (SUMO) both in vitro and in transfected redundancy. However, some important non-redundant functions of cells.65 This finding is potentially interesting, since accumulating evid- Peli1 have recently been uncovered through the study of Peli1 KO ence suggests crosstalks between sumoylation and ubiquitination.66 mice.9,10 On the one hand, SUMO may interfere with ubiquitination by com- peting with ubiquitin for attaching to the same lysine residues. On the Regulation of TRIF-dependent TLR signaling other hand, sumoylation can prime certain proteins for ubiquitina- The Peli1 KO mice are grossly normal and do not have prominent tion. Mutagenesis analyses identified five lysine residues (K202, K266, abnormalities in the development of immune organs and cells.9 K295, K297 and K303) as potential SUMO acceptor sites of Peli1. Of However, these mutant animals display reduced sensitivity to endotoxin note, two of these residues (K202 and K266) are also used as ubiqui- shock in an acute inflammation model that involves injection of a low- tination acceptor sites, suggesting the possible involvement of direct dose of LPS together with D-galactosamine (a liver-specific translation crosstalks between sumoylation and ubiquitination in the regulation inhibitor that increases LPS sensitivity).9 In this model, the lethality is of Peli1 function. However, the functional significance of Peli1 primarily mediated through LPS-induced production of TNF-a and sumoylation has not been investigated. It also remains to be examined also involves other proinflammatory cytokines such as IL-6 and IL- whether this modification of Peli1 is induced during TLR/IL-1R sig- 12. The reduced lethality of Peli1 KO mice suggests impaired inflam- naling or the activation of other cellular pathways. matory responses to LPS. Indeed, the serum level of proinflammatory cytokines is greatly (TNF-a and IL-6) or moderately (IL-12) reduced in Transcriptional regulation the mutant animals upon LPS/D-galactosamine challenge. The Peli1 Another frequently seen mechanism of E3 regulation is through the deficiency also diminished the in vivo response of mice to polyIC injec- control of gene expression. An early study identified Peli1 as one of the tion in both cytokine production and lethality. In vitro studies further TRIF-dependent genes induced by the TLR4 ligand LPS in dendritic demonstrate a role for Peli1 in mediating LPS- and polyIC-induced cells.67 This finding has been confirmed and extended by a recent work proinflammatory gene expression in several cell types, most promi- demonstrating that Peli1 is induced at both RNA and protein levels in nently B cells and MEFs.9 LPS and polyIC stimulate TLR4 and TLR3, response to stimulation by both LPS and the TLR3 ligand polyIC.12 respectively, which are known to induce the TRIF-dependent TLR These TRIF-dependent TLRs are known to activate the IKK-related pathway leading to the induction of both proinflammatory cytokines

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and type I IFNs.25 However, Peli1 is dispensable for IFN induction by both LPS and polyIC, suggesting the selective involvement of Peli1 in the proinflammatory axis of TRIF-dependent TLR signaling. One major surprise in the Peli1 KO mouse study is the finding that Peli1 is dispensable for IL-1-induced gene expression and signaling, since in vitro studies suggest a critical role for Peli family members in the regulation of IRAKs and IL-1R signaling. Peli1-deficient MEFs do not show any notable defect in IL-1-stimulated expression proinflammatory genes.9 Similarly, the Peli1 KO and control mice produce comparable levels of IL-6 in response to IL-1 injection. One possible explanation to these findings is that Peli1 may be functionally redundant with other Peli1 members in the regulation of IRAKs. IRAK1 and IRAK4 are also involved in MyD88-dependent TLR signaling pathway. However, Peli1 appears to be largely dispensable for gene induction by the MyD88-dependent TLRs. This conclusion is not absolute, though, since the Peli1 deficiency does affect the function of some MyD88- dependent TLRs, such as TLR9 and TLR2/6, depending on the cell types.9 Of note, IRAK1 deficiency also does not cause an overall defect in the MyD88-dependent TLR signaling, although it affects some specific aspects of TLR signaling.71,72 Thus, another possibility is that Peli1 is required for the activation of IRAK1, but not that of IRAK4. The TRIF-dependent TLR pathway activates two major downstream signaling axes, which lead to activation of NF-kB and IRF3, respectively. As discussed already, the IRF3 activation is mediated by the IKK-related kinases, TBK1 and IKKe. An important signaling Figure 2 Peli1 mediates IKK activation by the TRIF-dependent TLR pathway. factor that connects TRIF-dependent TLR signals to NF-kB is the Based on the signaling adaptors, TLR signaling can be classified into two major 73 adaptor molecule RIP1. The signaling function of RIP1 involves its pathways: the MyD88- and TRIF-dependent pathways. TLR3 is the only TLR that conjugation of K63-linked polyubiquitin chains, which appear to exclusively signals through TRIF, whereas TLR4 signals through both TRIF and facilitate the recruitment and activation of downstream kinases, MyD88. The rest of the TLRs, as well as IL-1R, signal through MyD88. An import- Tak1 and IKK.26 Interestingly, the polyIC-induced RIP1 ubiquitina- ant mechanism of TLR signaling is K63 type ubiquitination of signaling adaptors, tion is largely blocked in Peli1-deficient MEFs.9 Consistently, Peli1 is including TRAF6, RIP1 and TRAF3, which mediate activation of downstream signaling events. Activation of IKK/NF-kB by the TRIF-dependent TLR pathway, required for polyIC-stimulated activation of IKK and NF-kB MEFs but not that of the MyD88 pathway, depends on Peli1. Peli1 functions by medi- and B cells. On the other hand, the Peli1 deficiency only partially ating ubiquitination of RIP1 and, thereby, activating IKK and NF-kB. The TRAF3- inhibited the activation of IKK and NF-kB by LPS, reflecting the fact regulated signaling pathway may crosstalk with the RIP1 pathway through induc- that TLR4 also mediates IKK/NF-kB activation via the MyD88- tion of Peli1 gene expression and TBK1-/IKKe-mediated Peli1 activation. IKK, IkB dependent TLR pathway. Furthermore, Peli1 is dispensable for IKK/ kinase; Peli1, Pelle-interacting protein 1; RIP1, receptor-interacting protein 1; TCR, T-cell receptor; TLR, Toll-like receptor. NF-kB activation by IL-1R and MyD88-dependent TLRs, further emphasizing a role for Peli1 in regulating the TRIF-dependent TLR signaling (Figure 2). It is important to note, however, the phenotype of revealed an unexpected role for Peli1 in regulating T-cell activation Peli1 deficiency in the TRIF-dependent TLR signaling varies among and homeostasis.10 This finding was triggered by a gene expression cell types, which is prominent in B cells and MEFs and only moderate analysis to compare the relative expression levels of different Peli family 9 in bone marrow-derived macrophages and dendritic cells. Clearly, members in innate and adaptive immune cells. Notably, both innate more work is warranted to examine the potential functional redund- immune cells (macrophages and dendritic cells) and MEFs express at ancy of the Peli family members in both the IL-1R and TLR signaling least two of the three Peli members at comparable levels, further indi- pathways. While generation of triple KO mice, lacking all three Peli cating potentially functional redundancy of Peli in certain aspects of members, is the ultimate goal, another approach is to find the cell types TLR/IL-1R signaling. Interestingly, however, Peli1 is unique among the that predominantly express Peli1 over the other Peli members. As will family members in that it is highly expressed in B and T lymphocytes. be discussed below, this approach has led to the discovery of an unex- Although B cells also have substantial expression of Peli3, T cells only 10 pected function of Peli1 in the regulation of T-cell activation. More express a negligible level of Peli2 and Peli3 compared to the strikingly comprehensive gene expression analyses may lead to the identification high level of Peli1 expression.10 Moreover, the level of Peli1 is further of relevant cell types for the study of TLR/IL-1R signaling. elevated following T-cell activation. These findings suggest a role for Nevertheless, the current findings suggest that Peli1 mediates RIP1 Peli1 in regulating T-cell activation. ubiquitination and plays a critical role in mediating NF-kB activation Based on the critical role of Peli1 in mediating TLR signaling, it by TRIF-dependent TLR pathway (Figure 2). would predict that loss of Peli1 in T cells might impair T-cell activation and cause immune deficiency. Surprisingly, the opposite function of Regulation of T-cell activation and autoimmunity Peli1 was discovered using the Peli1 KO mice.10 The Peli1-deficient T As discussed above, both in vitro and in vivo studies have established cells are hyper-responsive to in vitro stimulation by agonistic TCR/ Peli as a family of innate immune regulators. However, since Peli pos- CD28 antibodies (anti-CD3 and anti-CD28) or T-cell mitogens (phor- sesses both K63 and K48 E3 ligase activities, it suggests the involvement byl myristate acetate and ionomycin). The mutant T cells appear to of Peli in different biological functions. Indeed, a recent study has also undergo activation in vivo, since the Peli1 KO mice contain

Cellular & Molecular Immunology Peli: immunoregulatory E3 ubiquitin ligases W Jin et al 119 substantially increased frequencies of CD4 and CD8 T cells with mem- tolerance. This finding is consistent with the hyper-responsive pheno- ory or effector phenotypes. Under normal conditions, T cells are tightly type of the Peli1-deficient T cells to in vitro stimulation. controlled so that they are responsive to foreign antigens, but are Loss of Peli1 does not substantially influence the TCR-proximal tolerant to antigens derived from self-tissues or commensal microbes.74 signaling events, such as activation of the protein tyrosine kinase Since the animals are housed under specific pathogen-free conditions, Zap70, or the activation of downstream kinases, IKK and ERK. The the in vivo T-cell activation in Peli1 KO mice is very likely due to the early phase activation of NF-kB, following T-cell activation by ago- loss of peripheral T-cell tolerance. Indeed, the activated T cells seem to nistic anti-TCR and anti-CD28 antibodies or mitogens, is also largely be self-reactive and infiltrate into various nonlymphoid organs and normal in the Peli1-deficient T cells. However, the Peli1 deficiency cause tissue inflammation. At old ages, the Peli1 KO mice also have results in aberrant activation of late-phase NF-kB. This is due to a other symptoms of autoimmunity, including elevated antinuclear specific role for Peli1 in the negative regulation of c-Rel, an NF-kB autoantibodies and immunoglobulin deposition in kidney glomeruli.10 family member that is activated with delayed kinetics and is critically Peripheral T-cell tolerance is mediated by both intrinsic and involved in T-cell activation.79 Peli1 appears to negatively regulate c- extrinsic mechanisms; the intrinsic mechanism involves negative Rel by mediating the ubiquitination and proteolysis of activated c-Rel regulation of the TCR/CD28 signaling by T-cell intrinsic molecules, (Figure 3). In response to TCR/CD28 signals, c-Rel undergoes degra- whereas the extrinsic mechanism involves the immunosuppressive dative ubiquitination in wild-type T cells, which was detectable only function of T regulatory (Treg) cells and other suppressive cells.75–78 when the cells were incubated with a proteasome inhibitor. This Thus, autoimmunity may occur due to either Treg defect or impaired molecular event is dependent on Peli1. At least in a transfection model, negative regulation of TCR/CD28 signaling. Peli1 is not required for Peli1 induces c-Rel ubiquitination with K48 linkage. It remains to be the generation or in vitro suppressive function of Treg cells. Although further examined whether Peli1 functions as the direct E3 of c-Rel it is unclear whether Peli1 mediates the in vivo function of Treg cells, or indirectly induces c-Rel ubiquitination through another E3. mixed bone marrow adoptive transfer study clearly demonstrates Nevertheless, Peli1 physically interacts with c-Rel under both a role for this E3 in mediating the T-cell intrinsic mechanism of endogenous and transfected conditions. Controlling c-Rel activation

Figure 3 Peli1 negatively regulates T-cell activation by mediating c-Rel degradation. In T cells, c-Rel is an important NF-kB member that regulates IL-2 gene expression and T-cell activation. c-Rel functions as either an homodimer or heterodimers associated with different NF-kB members, such as p50 and RelA. Upon IKK- mediated degradation of IkB, the c-Rel-NF-kB dimers translocate to the nucleus, where they participate in the transactivation of IL-2 and other target genes. The TCR/ CD28 signals also stimulate the expression of Peli1 via a yet-to-be-characterized transcription factor(s), and the accumulated Peli1 negatively regulates the late-phase activation of NF-kB by mediating ubiquitination and degradation of c-Rel. It is currently unknown whether Peli1 exerts this function in the cytoplasm or nucleus, but Peli1 is located in both of these cellular compartments (Chang M et al., unpubl. data). IKK, IkB kinase; Peli1, Pelle-interacting protein 1; RIP1, receptor-interacting protein 1; TCR, T-cell receptor; TLR, Toll-like receptor.

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may be an important mechanism by which Peli1 mediates intrinsic T- factors. In particular, the characterization of Peli members as RING cell tolerance, since c-Rel is crucial for the activation and differenti- finger E3s has provided critical clues to their signaling functions. Since ation of T cells and for the prevention of T-cell tolerance.80–86 Peli possesses both K63 and K48 types of ubiquitin ligase activity, the function of Peli may be more complex than what was initially pro- Interplay with TGF-b signaling components posed. Indeed, recent Peli1 gene-targeting studies have revealed critical, In addition to directly manipulating the key molecules in NF-kB sig- but opposing, functions in the regulation of innate immunity and T- naling cascade, Peli1 can also mediate crosstalk between the NF-kB cell activation. One future task is to characterize additional physio- and TGF-b pathways, which are known to antagonize each other logical or pathological functions of Peli and to examine how the under inflammatory settings.87–89 TGF-b belongs to a superfamily of different functions of Peli are regulated. Since Peli members share cytokines, which also includes bone morphogenic proteins and acti- extensive sequence homology, it is important to examine whether vin. TGF-b is widely expressed in immune cells and plays unique and the different Peli proteins have functional redundancies. Another pivotal regulatory roles in the regulation of immune responses.90,91 important issue to be addressed is the potential association of Peli with The TGF-b signaling pathway involves sequential activation of the human diseases. Although this possibility is suggested by the current receptor complex (type I and II receptors) and the signal transducers, findings, it is yet to be examined by genome-wide association studies. Smads, upon receptor-ligand binding.87,92 There are eight mam- Understanding how Peli is regulated in vivo represents another malian Smads, which can be subgrouped as receptor-activated challenge for future studies. Nevertheless, an important clue has been Smads, common Smad and inhibitory Smads (I-Smads) according provided by the recent observations that IRAKs and TBK1/IKKe medi- to their function. Smad2 and Smad3 are the two receptor-activated ate the inducible phosphorylation of Peli1 by IL-1R and TLR, respec- Smads selectively activated by TGF-b signaling, which form a complex tively. It appears that signal-induced Peli phosphorylation may serve with the common Smad, Smad4, and corporate with other transcrip- as a molecular trigger for its activation. A number of outstanding tion factors or cofactors to regulate downstream gene expression. The questions remain to be addressed in this area. For example, it is unclear TGF-b signaling pathway is tightly controlled by the I-Smads, Smad6 how Peli1 phosphorylation stimulates its E3 ubiquitin ligase activity. It and Smad7, which are induced in response to TGF-b stimulation and, remains to be tested whether phosphorylation promotes the binding thereby, form a feedback regulatory loop.92–95 Smad6 and Smad7 of Peli1 by specific E2s. Future studies should also examine whether attenuate TGF-b signaling by interacting with TGF-b receptor I, thus phosphorylation similarly regulates the E3 ligase activity of other Peli interfering with Smad2/3 phosphorylation and subsequent activa- family members in vivo and whether this involves the same or different tion.96,97 The I-Smads also recruit ubiquitin ligases to TGF-b receptor protein kinases. Another outstanding issue to be further addressed is I, leading to the polyubiquitination and degradation of the receptor.98 regarding the IRAK1/Peli mutual regulation model previously pro- The crosstalk between TGF-b signaling and other signal transduc- posed based on in vitro studies. Recent studies support the involve- tion pathways is at least partially mediated by the I-Smads. Multiple ment of IRAK1 in the in vivo activation of Peli1, but the inverse inflammatory cytokines, such as IFN-c, TNF-a and IL-1b, induce function of Peli in IRAK1 regulation remains to be speculative. Smad7 protein expression and, thereby, counteract on the immuno- Notwithstanding, accumulative findings have established Peli as a suppression controlled by TGF-b signaling.99,100 It has also been new family of RING finger E3s with important roles in the regulation observed that TGF-b signaling antagonizes NF-kB activation induced of both innate immune receptor signaling and T-cell tolerance. by proinflammatory cytokines. In this regard, recent studies indicate that association between I-Smads and Peli1 is critical for attenuating ACKNOWLEDGEMENTS IL-1R-/TLR-induced NF-kB activation by TGF-b.88,89 It appears that The research of S-CS is supported by grants from the US National Institutes of TGF-b induces the expression of both Smad6 and Smad7, which Health (AI057555, AI064639 and GM84459) and the GS Hogan Gastrointestinal Cancer Fund. compete with IRAK1 and IRAK4 for engaging Peli1, thereby interfering with the formation of the MyD88/IRAK4/IRAK1/Peli1/TRAF6 signaling complex required for IKK/NF-kB activation. Indeed, the interaction of Smad6/7 with Peli1 blocks IL-1R-/TLR-stiumulated 1 Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem 1998; 67: 425– IkBa degradation, NF-kB nuclear translocation and subsequent 479. induction of inflammatory genes in both HEK293 cells and primary 2 Chen ZJ, Sun LJ. Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell macrophages.88,89 Both the induction of I-Smads and their physical 2009; 33: 275–286. 3 Sun SC. Deubiquitylation and regulation of the immune response. Nat Rev Immunol interactions with Peli1 are required for TGF-b-mediated negative 2008; 8: 501–511. regulation of NF-kB. The MH2 domain of I-Smads is responsible 4 Ardley HC, Robinson PA. E3 ubiquitin ligases. Essays Biochem 2005; 41: 15–30. for their association with Peli1, which has non-redundant binding 5 Li W, Bengtson MH, Ulbrich A, Matsuda A, Reddy VA, Orth A et al. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a sequences for Smad6 and Smad7, suggesting potentially simultaneous mitochondrial E3 that regulates the organelle’s dynamics and signaling. PLoS ONE interaction of Peli1 with both I-Smad proteins.89 Indeed, induction of 2008; 3: e1487. both I-Smads can suppress NF-kB activity more effectively than any 6 Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem 2009; 78: 399–434. one of the two. These findings shed light on a novel function of Peli1 7 Moynagh PN. The Pellino family: IRAK E3 ligases with emerging roles in innate in NF-kB regulation, although additional studies are required for immune signalling. Trends Immunol 2009; 30: 33–42. better understanding the underlying molecular mechanism and 8 Schauvliege R, Janssens S, Beyaert R. Pellino proteins: novel players in TLR and IL-1R signalling. J Cell Mol Med 2007; 11: 453–61. physiological relevance of the Peli1/I-Smad interplay. 9 Chang M, Jin W, Sun SC. Peli1 facilitates TRIF-dependent Toll-like receptor signaling and proinflammatory cytokine production. Nat Immunol 2009; 10: 1089–1095. 10 Chang M, Jin W, Chang JH, Xiao Y, Brittain GC, Yu J et al. The ubiquitin ligase Peli1 CONCLUSIONS AND OUTSTANDING QUESTIONS negatively regulates T cell activation and prevents autoimmunity. 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