How Do Pleiotropic Kinase Hubs Mediate Specific Signaling by TNFR

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Ba¨rbel Schro¨felbauer How do pleiotropic kinase hubs Alexander Hoffmann mediate speciﬁc signaling by TNFR superfamily members?

Authors’ address Summary: Tumor necrosis factor receptor (TNFR) superfamily mem- 1 1 Ba¨rbel Schro¨felbauer , Alexander Hoffmann bers mediate the cellular response to a wide variety of biological inputs. 1 Signaling Systems Laboratory, Department of Chemistry and The responses range from cell death, survival, differentiation, prolifera- Biochemistry, University of California, San Diego, La Jolla, tion, to the regulation of immunity. All these physiological responses are CA, USA. regulated by a limited number of highly pleiotropic kinases. The fact that the same signaling molecules are involved in transducing signals from Correspondence to: TNFR superfamily members that regulate different and even opposing Ba¨prbel Schro¨felbauer processes raises the question of how their specificity is determined. Regu- Signaling Systems Laboratory latory strategies that can contribute to signaling specificity include scaf- Department of Chemistry and Biochemistry folding to control kinase specificity, combinatorial use of several signal University of California, San Diego transducers, and temporal control of signaling. In this review, we discuss NSB 0375 these strategies in the context of TNFR superfamily member signaling. 9500 Gilman Dr La Jolla, CA 92093-0375, USA Keywords: TNFR, MAPK, IKK, NFjB, scaffold, signaling specificity Tel.: +1 858 822 4670 Fax: +1 858 822 4671 e-mail: [email protected] Diverse biological responses to TNFR superfamily members Acknowledgements This work was supported by RO1 grant CA141722. BS is a The tumor necrosis factor receptor (TNFR) superfamily Leukemia and Lymphoma Society fellow. The authors have consists of 29 receptors that mediate cellular responses to 19 no conflicts of interest to declare. ligands. While most ligands bind to a single receptor, some bind to numerous receptors. For example, BAFF can associate with three receptors, and TNF-related apoptosis-inducing This article is part of a series of reviews ligand (TRAIL) can even bind to five receptors (1, 2). All TNFRs covering TNF Receptor Family Members are characterized as type I transmembrane proteins, with an appearing in Volume 244 of Immunological Reviews. extracellular N-terminus and intracellular C-terminus necessary for signaling initiation (3). The TNFR superfamily can be cate- gorized into three overlapping classes: activating receptors, death receptors, and decoy receptors. Activating receptors such as TNFR1 and CD40 mediate activation of nuclear factor-jB (NFjB) and mitogen-activated protein kinase (MAPK) pathways. Death domain containing receptors (such as TNFR1 and FAS) contain an 80 amino acid death domain in their cytoplas- Immunological Reviews 2011 mic domain. Its deletion abolishes ligand-induced cell death. Vol. 244: 29–43 Printed in Singapore. All rights reserved Through sequestration of the ligand, decoy receptors (e.g. DCR1, OPG) have been shown to inhibit cell signaling. 2011 John Wiley & Sons A/S Immunological Reviews Most receptors are expressed on a wide variety of cell types. 0105-2896 Receptor engagement by members of the TNF superfamily

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can trigger diverse cellular responses, such as apoptosis [for Pleiotropic signal transducers within the TNFR signaling example TNF, lymphotoxin (LT), FAS ligand (FASL)], survival network [receptor activator of NFjB ligand (RANKL) and B-cell activat- The key signal transducers of the TNFR superfamily members ing factor belonging to the TNF family (BAFF)], differentia- are the kinases IKK2, IKK1, JNK, p38, and ERK. Below we tion (such as TNF, RANKL), or proliferation (such as TNF, have summarized some key facts about each kinase, indicating CD40L, OX40L, BAFF). These cellular responses are mediated the wide variety of substrates and biological functions that by the activation of transcription factors NFjB, which com- have been ascribed to each (Fig. 1). prise NFjB1 (p50 and its precursor p105), NFjB2 (p52 and IKK2 its precursor p100), c-Rel, RelA (p65), and RelB, and activator protein-1 (AP-1), composed of Fos (c-Fos, FosB, Fra-1, and IKK2 is a key regulator of NFjB activation induced by Fra-2) and Jun (c-Jun, JunB, and JunD) family members, as inflammatory cytokines, pathogens, environmental and meta- well as closely related transcription factors CREB, ATF2, ATF3, bolic stress and some developmental signals. By phosphory- and B-ATF), which are activated by MAPK [p38, c-Jun N-ter- lating the classical IjBs (IjBa,IjBb,IjBe), it triggers their minal kinase (JNK), extracellular signal-regulated kinase proteasomal degradation and release of NFjB to the nucleus (ERK)] and inhibitor of NFjB kinase (IKK) signaling cascades to allow for transcriptional activation. The primary canonical (1, 2). NFjB effectors are RelA, cRel, and p50 in both homodimeric Signaling by TNF superfamily members is essential for a or heterodimeric forms. Induction of their activity by IKK2 large variety of physiological processes including hematopoie- leads, in most cell types, to a general cell activation response, sis, protection from bacterial infection, immune surveillance, that may involve the secretion of inflammatory molecules, and tumor regression. Ligands, including TNF, LTb, and increasing the resistance to cell death-inducing stimuli, and, RANKL, provide crucial signals for the morphogenesis of sec- in lymphocytes particularly, activation of a proliferative pro- ondary lymphoid organs; TNF, FAS, and TRAIL contribute to gram (7, 8). Apart from its role in activating NFjB, IKK2 has the function of cytotoxic effector cells in the recognition and many additional substrates that are not part of the NFjB sig- destruction of virus-infected cells. The expression of FASL on naling system. The IKK2-dependent phosphorylation of the activated T cells induces their cell death, a mechanism to mod- tumor suppressor p53 at S362 and S366 is thought to be a ulate the immune response. Importantly, misregulation of mechanism for regulating its stability (9). Upon TNFa stim- TNFR signaling has been associated with a diverse range of ulation, IKK2 phosphorylates insulin receptor (IR) substrate diseases including autoimmunity, liver disease, tumorigenesis, 1 (IRS-1) at S307, resulting in the termination of metabolic lymphproliferative diseases, diabetes, and even allergic asthma insulin signaling (10). In addition, IKK2 phosphorylates the (1, 2, 4–6). The diverse pathological effects caused by TNFR tumor suppressor tuberous sclerosis 1 (TSC1) at position misregulation reflect the large variety of biological processes S487 and S511, which leads to its suppression and coincides they are involved in and highlight the importance of precise with the activation of the mTOR pathway, enhanced angio- regulation of TNFR superfamily signaling. Interestingly, the genesis and tumor development (11). b-catenin, a key mole- MAPK and IKK cascades are the critical signal transducers for cule in Wnt signaling, and FOXO3a, which acts downstream all TNFRSFs raising the question of how the functional speci- of growth factor signaling (PI3K ⁄ Akt), have also been ficity of these kinases is determined to ensure signaling speci- reported to be specifically phosphorylated by IKK2 (12). ficity. Recently, IKK2 has also been found to play a critical role in

Fig. 1. Kinases involved in TNFRSF-member signaling are highly pleiotropic. The pleiotropic kinases JNK, p38, and ERK1 ⁄ 2 (MAPKs), canonical IKK2, and non-canonical IKK1 are key regulators in signal transduction in response to a large variety of cellular signals and can trigger highly diverse biological responses.

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AB C

Fig. 2. Activation of signal transducers downstream of TNFR1, CD40, and LTbR engagement. (A) TNF triggers the assembly of a signaling complex involving the TRADD, RIPK1, and ubiquitin ligase complexes. TAK1 is recruited and activated by binding of its scaffolds TAB 2 ⁄ 3 to polyubiquitin chains. Subsequent binding of NEMO to ubiquitin chains allows for the activation of IKK2. MAP3Ks activate MAPKs through a cascade of phosphorylation events. (B) CD40 engagement triggers the assembly of a complex containing TRAF2, TRAF3, TRAF6, cIAPs, MEKK1, and the LUBAC complex to activate MAPKs, NEMO-IKK2, and non-canonical IKK1. (C) Binding of LTb to the LTbR triggers degradation of TRAF2 and TRAF3 resulting in NIK stabilization, which activates non-canonical IKK1 (see text for details). starvation-induced autophagy, in an NFjB independent thought to regulate cell proliferation in an NFjB-independent manner (13, 14). The substrates however remain to be manner (16–18). identiﬁed. JNK IKK1 JNK is a member of the MAPK family of signaling proteins The IKK1 kinase functions as a homodimer and as a heterodi- (19, 20). It consists of 10 JNK isoforms, which are derived mer with IKK2. As a homodimer it may be activated through through alternative splicing of mRNA transcripts generated the NFjB-inducing kinase (NIK) by developmental signals from the three genes JNK1, JNK2, and JNK3. It is activated in such as BAFF and LTb (6, 8). IKK1 dependent phosphoryla- response to cellular stress, cytokines, pathogens, and mito- tion of p100 results in p100 processing and induction of tran- gens. JNK activation leads to the phosphorylation of a large scriptional activation. The main mediators of transcription are number of transcription factors, the most prominent of which RelA, RelB, p50, and p52 controlling cell survival and devel- is c-Jun, a component of AP-1, as well as of numerous cellular opmental processes (6, 8). Similar to IKK2, IKK1 has also proteins most of which are associated with apoptosis (such as been reported to have additional NFjB independent sub- Bcl2 and p53). Gene disruption in mice revealed its essential strates. Like IKK2, IKK1 also phosphorylates b-catenin (15). role in TNFa-induced c-Jun phosphorylation and AP-1 tran- Interestingly, while IKK2-induced phosphorylation negatively scription factor activity. JNK is activated through phosphory- regulates b-catenin activity, phosphorylation by IKK1 lation of the Thr-Pro-Tyr motif in its activation loop by MAPK increases b-catenin-dependent transcription, indicating dis- kinases (MAPKKs) MKK4 and MKK7. Activation of these tinct biological roles for these kinases. Many other IKK1 sub- kinases in turn is initiated by a cascade of kinases linked strates localize to the nucleus, including the coactivators through stimulus-dependent association with different scaf- SMRT, SRC3, and CBP, as well as the histone H3, and they are fold proteins (see below). The complex regulation of JNK is

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indicative of its critical role in multiple physiological pro- ERK cesses. Indeed, JNK has been implicated in the regulation of cell survival and apoptosis, inflammation, metabolism, and ERKs are encoded by two genes, ERK1 (MAPK3), and ERK2 development. By phosphorylating several pro-apoptotic mem- (MAPK1), that encode two main proteins, p44 (ERK1), and bers of the Bcl2-related protein family (Bim, Bmf, Bad) and p42 (ERK2). They are preferentially activated by mitogens through JNK-dependent activation of AP-1, it can activate the but, similar to JNK and p38, are also activated in response to mitochondrial apoptotic pathway, while its phosphorylation GPCR agonists, cytokines, and environmental stress. Activation ⁄ ⁄ of several anti-apoptotic members (Bcl2, Bcl-xL, Mcl-1) pro- occurs through the Raf MEK ERK signaling cascade through mote cell survival. In addition, JNK has been implicated to different isoforms of the small GTP-binding protein Ras. Acti- play important roles in other types of death including necrosis vated Raf binds to and phosphorylates MEK1 and MEK2, ⁄ ⁄ and autophagic ⁄ lysosomal cell death. JNK is also a potent which in turn phosphorylate ERK1 2. Markedly, ERK1 2 have inducer of inflammatory gene expression primarily through been reported to phosphorylate more than 160 substrates, the activation of the transcription factors AP-1 and ATF-2. including numerous important transcriptional regulators such Accordingly, many autoimmune diseases (rheumatoid arthri- as NF-AT, Elk-1, c-Fos, c-Myc, and H3 to control the cell cycle tis, multiple sclerosis, psoriasis) correlate with hyperactivity progression and cell death. Apart from ERK’s critical role in of JNK. The activation of JNK causes insulin resistance at least cell proliferation, it is also critically involved in differentia- in part through the phosphorylation of Insulin receptor sub- tion, migration, and cellular transformation (28, 29). strate-1 (IRS-1) at the inhibitory site Ser-307, thus suppress- ing insulin receptor signaling, indicating its role in Activation of signal transducers downstream of specific TNFRSF members metabolism (19–21). The key kinases are connected to members of the TNFR super- p38 family via complex signaling mechanisms that involve a multitude of protein adapters and enzymatic functions. Their p38 is another member of the MAPK family. It consists of cooperative or sequential regulation control dose response and four isoforms, p38a, p38b, p38c, and p38d, each encoded dynamic behavior of the key kinases. Therefore, knowledge by a distinct gene. They have similar in vitro substrate speci- about the signaling molecules involved in the activation of the ficity but differ in regards to how their activity is regulated key signaling kinases and their regulation is of major impor- or their responsiveness to ligands; the p38 pathway is acti- tance to gain insights into how signaling specificity may be vated by a large number of growth factors, GPCR agonists, achieved. Below we detail what is currently known about these environmental stresses, pathogens, and cytokines. Dependent signaling mechanisms for three TNFRSF members (Fig. 2). on the isotype, they are activated by dual kinases (threonine and tyrosine) MKK3, and the JNK kinases MKK4 and MKK7. TNFR1 Once activated, p38 phosphorylates several cytoplasmic substrates (including MBP, HSP27, and MAPKAPK2) and upon Activation of TNFR1 triggers a wide variety of biological nuclear translocation activates the transcription factor ATF-2 responses from cell death to survival, effects on metabolism, through direct phosphorylation (22–24) as well as CREB differentiation, adhesion, and motility. TNFR1 is particularly (25). p38 can also indirectly regulate transcription through important in inflammatory responses (2). TNFR1 knockout AP-1. One of the main consequences of p38 activation is mice show impaired clearance of bacterial pathogens and increased expression of cytokines and receptors involved in resistance to LPS (30–34). Although lymph nodes develop inflammation and immunity (19, 21, 26). However, it normally, these mice show defects in germinal center forma- remains unclear whether the aforementioned transcription tion and impaired development of Peyer’s patches, indicating factors account for p38’s function in gene expression or a critical role of TNFR1 in acute immune responses (30, 31). another reported p38 function in stimulus-induced mRNA TNFR1 mediates its physiological functions through activation stabilization (27). In addition, p38 is involved in inflamma- of canonical IKK, JNK, MAPK, and ERK. tion, cell death, cell cycle regulation, and cell differentia- Upon binding of TNF to the TNF receptor, the first steps in tion. Inhibition of p38 using specific pharmacological signaling are the recruitment of the adapter molecules TRADD inhibitors results in reduced proliferation and cell cycle and RIPK1 and then of TRAF2 ⁄ 5 to TRADD (35). TRADD is arrest (19, 21, 26). essential for TNF-induced signaling in MEFs as in TRADD-

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deficient MEFs TNFR-induced IjBa phosphorylation and deg- replace TAK1 or whether they act cooperatively is currently radation are completely abolished (36–38). In contrast, in not clear. Activation of the NEMO-IKK complex results in macrophages the TRADD requirement is less complete, sug- phosphorylation and degradation of the canonical IjBs and gesting that in these cells TRAF2 may be recruited in a NFjB activation. TRADD-independent manner (38). The requirement for RIP1 In addition to the activation of IKK, TNFR1 also leads to the varies between cell types; while largely dispensable in MEFs potent activation of JNK, p38, and ERK1 ⁄ 2. Activation of JNK (39–41), it is absolutely required for TNF-induced NFjB acti- depends on TRAF2 and on the formation of ‘non-destructive’ vation in T and B cells, as human T cells lacking RIPK1 and K63 ubiquitin chains (63–70). The involvement of linear pre-B-cell lines derived from RIPK1 knockout mice show chains is still controversial (53, 54, 71). Activation of p38 defects in NFjB activity (42–44). Notably, its kinase activity similarly depends on TRAF2 and RIPK1; however, the contri- is not required (39). TRAF2-deficient mice show only a slight bution of ubiquitin chains is unclear (72, 73). Several reduction in TNFR-induced NFjB activation, while TRAF2 ⁄ 5 MAP3Ks have been implicated in the activation, including double deficiency results in complete impairment, suggesting TAK1 (74), MEKK1 (74), TPL-2 (75), and ASK1 (74, 76, 77). redundant functions for these molecules (39). However, this Genetic deletions of MAP3Ks have not revealed an absolute topic remains controversial. requirement for any kinase, indicating some redundancy in Subsequently, cIAP1 ⁄ 2 are recruited to the complex, which TRAF2-dependent JNK and p38 activation. MAP3Ks then is not only important for NEMO-containing IKK activation phosphorylate and activate MAP2Ks, which are somewhat (45–47) but also for inhibition of the non-canonical IKK1 pathway specific. While MKK3 and MKK6 specifically phos- activity, as these E3 ligases are also involved in constitutive phorylate and activate p38, MKK7 activates JNK. MKK4 is less degradation of NIK (48, 49) (see below). Within this com- specific and can activate both p38 and JNK (reviewed in 78). plex, RIPK1, TRAFs, and NEMO undergo several forms of The main activator of the TNF-induced ERK1 ⁄ 2 cascade is ‘non-destructive’ ubiquitination, primarily conjugation of TPL-2 (79). Macrophages from Tpl2 knockout mice are defec- K63 or head-to tail (linear) polyubiquitin chains. While K63 tive in ERK1 ⁄ 2 activation induced by TNF, while activation of ubiquitination of RIPK1 and NEMO is catalyzed by the E3 lig- other MAPKs and NFjB remain unaffected (79). In contrast, ases cIAP1 ⁄ 2 or TRAF2 together with Uev1A and Ubc13 (44, TNF stimulation of Tpl2-deficient MEFs results in defective 45, 50), the LUBAC complex, consisting of HOIL-1, HOIP, activation of JNK, p38, ERK1 ⁄ 2 as well as NFjB (75), indicat- and SHARPIN, exclusively modifies RIPK1 and NEMO with ing a cell type-specific role for TPL-2. linear polyubiquitin chains (51–54). Conjugation of K63 and linear ubiquitin chains to RIP1 triggers the recruitment of IKK activating kinases. First, the IKK-kinase TAK1 (IKK-K) is CD40 recruited by specific binding of its scaffolds TAB2 ⁄ 3 to the CD40 is expressed on DCs, B cells, and endothelial cells. In ubiquitin chains (39, 50, 55). TAK1 itself is K63 ubiquitinat- DCs, CD40 signaling promotes cytokine production, induced, which triggers its autophosphorylation and activation tion of costimulatory molecules, and facilitates cross presenta- (55–57). In vitro unanchored K63 polyubiquitin chains have tion; in B cells, CD40 signaling can promote germinal center been shown to be sufficient for TAK1 activation (58), but formation, isotype switching, somatic hypermutation, and whether conjugation of ubiquitin chains to components of formation of plasma cells and memory B cells. In addition, it the complex may contribute to dose-responsiveness or speci- has been demonstrated to be important for the survival of ficity in vivo remains to be investigated. many cell types, including GC B cells, DCs, and endothelial Binding of NEMO to these ubiquitin chains brings IKK in cells. CD40-deficient mice show defective B-cell development, close proximity to TAK1, allowing for IKK activation by Ig class switching, and GC formation, ultimately causing phosphorylation of the activation loop serines (50–52, 59). immunodeficiency (80–83). In similarity to TNFR1, CD40’s TAK1 appears to be the major kinase responsible for IKK acti- physiological functions are also mediated by the signal trans- vation, as TAK1-deficient cells are largely defective in TNF- ducers IKK1, IKK2, JNK, p38, and ERK. induced NFjB activation (60, 61). However, MEKK3 has also In contrast to TNFR1 signaling, activation of CD40 signal- been reported to act as an IKK kinase. Similar to TAK1, ing does not require the adapter TRADD but instead is initi- MEKK3 binds to RIPK1 and TRAF2 and can phosphorylate ated by binding of ubiquitin ligases TRAF2, TRAF3, and IKK2 (62). Consistently, MEKK3 deficiency impairs IKK and TRAF6 directly to the receptor (84, 85). In the case of CD40 MAPK activation in fibroblasts (62). Whether MEKK3 can activation, the TRAF molecules do not appear to have

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redundant functions. TRAF2 has been shown to be the pri- In similarity to TNFR1 signaling, both TAK1 and MEKK3 mary mediator of JNK and p38 activation (67, 68, 86). are implicated as the activating kinases for canonical IKK by Engagement of TRAF2 to CD40 results in the recruitment of CD40 ligand engagement (57, 106, 107). Several kinases have MEKK1 (87), which drives the phosphorylation of JNK and been suggested to mediate activation of MAPK pathways. As p38. TRAF2-deficient fibroblasts are defective in CD40L- mentioned above, MEKK1 is recruited to the CD40 complex induced JNK and p38 activation, with only little defect in through its interaction with TRAF2 to induce JNK and p38 NFjB activation (67, 68, 86). Attenuation of MAPK activation activation (87). Accordingly, in MEKK1-deficient B cells no was also observed in TRAF2-deficient B cells (88). In addi- JNK and p38 activation can be detected, while there are con- tion, TRAF2 deficiency caused constitutive p100 processing flicting results on the effects on NFjB activation (108–111). and elevated c-Rel activity (88). This reflects the fact that in Several other MAP3Ks have been suggested to be important contrast to TNFR1, CD40 engagement also triggers the activa- for JNK and p38 activation, including TAK1, MEKK3, and tion of non-canonical IKK through NIK (89). In unstimulated TPL-2. Although TAK1 is critical for TNF, BCR, and TLR cells, NIK expression is very low due to its constitutive degra- ligand induced activation of MAPKs, it appears to only play a dation by cIAPs, TRAF2, and TRAF3 (48, 49). CD40 engage- minor role in CD40-induced signaling, as CD40 engagement ment triggers self-degradation of TRAF2 and cIAP-dependent in TAK1-deficient B cells shows only modest defects in JNK degradation of TRAF3, resulting in stabilization and accumu- and p38 activation (61). In overexpression experiments lation of NIK (90–93). Indeed, germ line inactivation of MEKK3 has been demonstrated to be able to induce JNK and either TRAF2 or TRAF3 leads to NIK accumulation and con- p38 activation (112), due to embryonic lethality the physio- stitutive p100 processing, indicating that NIK accumulation is logical relevance for CD40-induced MAPK activation however sufficient for its activation of non-canonical NFjB pathway remains unclear (113). Interestingly, the MAP3K TPL-2 does (94–96). It subsequently activates IKK1 through phosphoryla- not appear to be involved in JNK, p38, or NFjB activation in tion of activation loop serines. NIK and IKKa phosphorylate CD40 stimulated B cells (79) but instead plays a critical role in p100 resulting in its processing and induction of NFjB tran- the activation of the ERK pathway (79). Tpl2-deficient mice scription (97–99). show a partial activation defect of ERK in response to CD40 Binding of TRAF6 to CD40 is important for activation of and TLR activation resulting in partial inhibition of IgE pro- JNK and p38 activation as well as NFjB (100). Its deficiency duction (79, 114). TPL-2 has also been implicated to be results in reduction or abrogation in the activation of canoni- involved in processing of NFjB1 p105 (115); however, pro- cal NFjB, JNK, and p38 (101, 102). Similar to TNFR signal- cessing appeared to be normal in Tpl2 knockout mice (79). ing, the cIAP1 ⁄ 2 and LUBAC ubiquitin ligase complexes have also been shown to be recruited to the CD40 receptor (103– 105). Whereas TNFR1 signaling involves conjugation of K63 LTbR and linear Ub-chains to recruit RIPK1 and NEMO and mediate LTbR signaling primarily controls the development of second- activation of TAK1 and downstream MAPK and canonical IKKs ary lymphoid organs (lymph nodes and Peyer’s patches). (44, 45, 50–54), CD40 signaling requires ubiquitination of LTbR-deficient mice lack all lymph nodes, Peyer’s patches, TRAF2 and TRAF6 for these downstream signaling events. and display a disturbed splenic architecture (116–118). In Interestingly, K63 ubiquitination appears to be critical for JNK adults, LTbR also controls the maturation and maintenance of and p38 activation and less important for CD40-induced the microarchitecture of lymphoid organs through expression ) ) NFjB activation, as B cells and macrophages from ubc13 ⁄ of specific chemokines (such as CXCL13 and CCL19), which mice, which are defective in catalyzing K63 chains, show induce stromal cell differentiation (117, 119). strong defects in JNK and p38 activation, while NFjB activa- In contrast to CD40 signaling, which leads to the activation tion is largely intact (105). In contrast, interfering with com- of MAPKs, the canonical NEMO-IKK2 complex and non- ponents of the LUBAC complex results in defective NFjB and canonical-NIK-IKK1, LTb, and BAFFR only induce NEMO- JNK activation (52, 54), indicating differential requirements independent, non-canonical IKK1. In similarity to CD40, the of Ub chains for MAPK and NFjB activation. A more detailed cytoplasmic domain of LTbR associates with TRAF3 and understanding of the nature of Ub-chains important for sig- TRAF2 (120, 121). Ligation of LTab or LIGHT to the LTb naling, the proteins they are conjugated to (if any) and the receptor triggers the degradation of TRAF2 and TRAF3 result- kinetics of Ub-chain formation will be important for a better ing in accumulation of NIK and subsequent activation of IKK1 understanding of signaling specificity. (see CD40 signaling). Through IKK1-induced degradation

34 2011 John Wiley & Sons A/S • Immunological Reviews 244/2011 Schro¨felbauer & Hoffmann Æ Signaling speciﬁcity of TNFRSFs

and processing of p100 RelA:p50 and RelB:p52 dimers are interactions with substrates, binding can trigger allosteric con- released into the nucleus to activate transcription (122–124). formational changes that can in turn affect strength and dura- NFjB activation induced by developmental signals such as tion of MAPK signaling (133). However, not all known LTb is considerably slower and weaker as compared to inflam- substrates have docking sites, suggesting that additional mech- matory signals. LTbR engagement also weakly induces JNK anisms are likely to exist in order to achieve substrate selectiv- activation in a TRAF2-dependent manner (125, 126); the ity to ensure efficient and specific signaling (127). exact mechanism however remains elusive. IKK1 and IKK2 are also known to be pleiotropic kinases that can phosphorylate a wide range of substrates influencing Mechanisms that ensure signaling specificity diverse cellular responses (134, 135). Interestingly, in in vitro assays IKKs do not show a high degree of substrate specificity, As described above, TNFRSF signal transduction pathways and in contrast to MAPKs, IKKs do not appear to have docking involve a small number of signaling enzymes, yet their bio- sites raising the question of how their activity is regulated to logical responses are highly diverse. Over the past few years, ensure signaling specificity. To understand the molecular basis many studies using a variety of genetic tools have revealed that for signaling specificity in cellular pathways, it will be of the functional requirements of specific signaling proteins in major importance to further elucidate mechanisms that con- signal transduction are cell type specific. Cell type-specific trol substrate specificity in vivo. genetic requirements may be the result of differential expression of parallel, potentially compensating pathway components. However, it may also be the result of cell-type specific Signaling specificity via scaffolding expression of proteins that modulate the function or kinetics When analyzing signaling responses to diverse biological of the key signaling enzymes that thereby control signaling inputs, it becomes clear that many signal transducers are specificity. shared, and, as described above, their substrate specificities Broadly, the specificity of signaling can be controlled at the appear limited. Yet, the biological outputs are highly specific. level of substrate specificity of the key kinases, differential Signaling specificity can be achieved by organizing discrete wiring of signaling inputs through scaffolding, the kinetics of subsets of proteins in space and time. One way to achieve this the kinase activities, as well as through combinatorial and is by sequestering functionally interacting proteins into temporal control mechanisms. Below we discuss some of specific subcellular compartments, such as organelles or the these mechanisms of regulation and how they may contribute plasma membrane. Another prevalent strategy is the assembly to signaling specificity. of functionally interacting proteins into specific complexes through protein scaffolds. Scaffolds bind to two or more com- Regulation of the enzymatic substrate specificity of kinases ponents of a cascade, bringing them in close proximity, MAPKs and IKKs are highly pleiotropic kinases, involved in thereby not only facilitating efficient propagation of the signal numerous distinct biological responses by phosphorylating a but also mediating its insulation from other signals. In addi- diverse array of substrates. MAPKs have been estimated to have tion to organizing signaling molecules into signalosomes, as many as 200–300 substrates each (127–129). One mecha- scaffolds can also have allosteric effects on the kinases thus nism by which specificity may be ensured in vivo may involve regulating kinase activity itself. co-factors that alter the intrinsic enzymatic specificity. How- The most prominent signaling cascades that are regulated by ever, unlike other enzymes such as prokaryotic RNA polyme- scaffolds are the MAPK pathways. As mentioned above, initia- rases whose DNA binding specificity is regulated by sigma tion of JNK and p38 pathways is triggered by many MAP3Ks factors, surprisingly little information has emerged about including TAK1, ASK1, MEKKs, and TPL-2 (74–77), which, specificity factors for the pleiotropic kinases in the TNFR sig- among others, can phosphorylate MKK4 and MKK7 to activate naling network. JNK, and MKK3, MKK4, and MKK6 to activate p38 (78). The MAPKs are known to have substrate binding sites, usually usage of alternative kinases at each step of the cascade might referred to as docking domains (130–132). These domains allow for the precise stimulus-dependent control of MAPKs. are distinct from the serine ⁄ threonine phospho-acceptor sites Indeed, the stimulus-specific organization of different kinases and consist of positively and negatively charged residues. into cascades by scaffolds can create functional signaling mod- Docking interactions themselves can be regulated by post- ules to control specificity of signal transduction (Fig. 3). For translational modifications (130–132). Apart from regulating example, filamin has been identified as a scaffold that may

2011 John Wiley & Sons A/S • Immunological Reviews 244/2011 35 Schro¨felbauer & Hoffmann Æ Signaling speciﬁcity of TNFRSFs

ABC

Fig. 3. Scaffolding to achieve signaling specificity. Scaffolds can organize pleiotropic kinases into stimulus-specific signaling modules to direct them to one specific pathway. (A) Filamin interacts with TRAF2, MEKK4, and JNK to allow for JNK activation upon TNFR1 engagement. ERK1 ⁄ 2 and p38 might similarly be organized into stimulus-specific modules through yet unidentified scaffolding proteins. (B) Binding of NEMO to IKK2 allows for its activation but is also required for proper signaling propagation to IjBa in response to inflammatory signals. (C) NIK is not only the activating kinase of IKK1 but also channels IKK1 activity to p100. coordinate the specific activation of JNK upon stimulation with 50–52, 55, 59). Thus, NEMO links upstream inflammatory TNF (136). The actin binding protein interacts with the signals to IKK2 through binding to ubiquitin chains. How is TNFR1 adapter TRAF2, the MAP2K MKK4, and JNK, thereby active IKK2 able to phosphorylate IjBs so efficiently and rap- coordinating a module that channels the upstream TNFR1 sig- idly while phosphorylation of alternative substrates is much nal to JNK (137). Accordingly, filamin deficiency causes a weaker and occurs with much slower kinetics? Our unpub- selective loss of TNF-induced JNK activation (136). The dual lished data suggest that IKK2-dependent phosphorylation of specificity protein phosphatase SKRP1 has also been reported IjB is also mediated by NEMO (BS and AH, unpublished to be involved in TNF-induced JNK activation through a scaf- data). Binding of NEMO to IjBs specifically channels IKK2 folding function facilitating the interaction of the MAP3K kinase activity towards inflammatory responses. Thus, scaf- ASK1, MAP2K MKK7, and JNK (138–141). Similar examples folding of NEMO ensures downstream signaling specificity by for a stimulus-specific organization of MAPK cascades by scaf- directing the pleiotropic IKK2 kinase to the canonical NFjB folds can be found for the activation of p38. The osmo-sensing pathway. The complex between NEMO-IKK2 and IjBa scaffold for MEKK3 (OSM) for example associates with Rac, appears to be constitutive, which might not only ensure MEKK3, and MKK3 to regulate p38 activation specifically in specific phosphorylation of IjBs but also enables the rapid response to osmotic shock (142). Other p38-specific scaffolds response, necessary for efficient inflammatory signal transduc- include JIP4 (143) and JLP1, which is implicated in p38- tion. In analogy to stimulus-specific MAPK kinase modules, dependent regulation of differentiation (144–146). Interest- ingly, the MAP3K MEKK1 not only acts as a kinase to activate JNK upon CD40 ligation, but itself possesses a scaffolding function by interacting with components of the JNK module (108, 147–151). It also constitutively interacts with Raf-1, MEK1, and ERK2 to facilitate stimulus-induced activation of ERK1 ⁄ 2 (147, 152, 153). TAK1, which is involved in transducing signals from diverse upstream receptors (e.g. TNFRs, TLRs, NLRs) to activate downstream JNK, p38, as well as NFjB pathways, associates with the scaffolds TAB1, TAB2, and TAB3. Apart from mediating TAK1 activation (see below), through binding to poly-ubiquitin chains, TAB2 ⁄ 3 also recruit TAK1 to NEMO-IKK2 to mediate NFjB activation. A well-known scaffold necessary for the activation of the Fig. 4. Combinatorial control of kinases facilitates stimulus specific- canonical IKKs is NEMO (154–156). Binding of NEMO to ity. Engagement of TNFRSFs activates pathways leading to the activation stimulus-induced polyubiquitin chains brings IKKs in close of ERK, JNK, p38, IKK2, and IKK1. Phosphorylation of downstream proximity to each other (allowing for trans-autophosphoryla- effectors induces a transcriptional program. Some genes require a synergistic function of several transcription factors and may thus be transcribed tion) and to the IKK-kinase TAK1, which then activates IKK2 in a stimulus-specific manner. Specificity can also be achieved post- through phosphorylation of its activation loop serines (39, transcriptionally through p38-dependent regulation of mRNA stability.

36 2011 John Wiley & Sons A/S • Immunological Reviews 244/2011 Schro¨felbauer & Hoffmann Æ Signaling speciﬁcity of TNFRSFs

NEMO assembles an inflammation-specific IKK module by results in defective JNK activation and shifts the balance of linking upstream IKK activators with the downstream effector signaling towards non-canonical IKK, despite a defect in IjBa. The specificity of IKK1 for p100 also seems to be regu- TRAF3 degradation (86, 88). This observation highlights the lated through scaffolding. NIK forms a complex with IKK1 importance of TRAF2 in negatively regulating the activation and p100, thereby not only activating IKK1 but also directing of non-canonical IKKs. In B cells, TRAF3 deficiency similarly IKK1s kinase activity towards p100. Thus, active IKK1 will results in enhanced activation of NIK-IKK but also in enhanced only induce p100 processing when NIK is stabilized by spe- canonical IKK and JNK activity (48, 49, 159, 160). Thus, cific developmental signals (e.g. LTb, CD40) (99). although both TRAF2 and TRAF3 are important negative regu- In summary, pathway-specific scaffolds can direct a pleio- lators of non-canonical IKK, they have different functions in tropic kinase to one specific pathway and at the same time regulating JNK activity. TRAF6 is dispensable for TNFR1 and prevent the activation of pathways irrelevant to that particular LTb signaling but is essential for CD40-induced JNK, p38, stimulus. Thus, scaffolds ensure stimulus-specific functions of ERK1 ⁄ 2, and canonical IKK activation (84, 85). kinases. The identification of scaffolds that are specific for each For activation of downstream kinases, TRAFs do not just act TNFR superfamily member will be critical to gain a better as adapters or scaffolds, but their E3 ubiquitin ligase activity is understanding about the regulation of signaling specificity. required. In conjunction with Uev1A and Ubc13, TRAFs mediate the conjugation of K63-linked ubiquitin chains onto other TRAFs, RIPK1, and NEMO, which has been shown to be Combinatorial coding critical for activation of NEMO-IKK2 and JNK by TNF as well Signaling specificity may also be mediated through the combi- as CD40L (44, 50, 70, 103–105). In fact, non-destructive natorial use of several pleiotropic signal transducers (Fig. 4). ubiquitin chains (such as K63 and linear chains) might add an In this scenario, stimulation of a single receptor triggers the additional level of regulation to achieve signaling specificity. formation of a signalosome that allows for the activation of a The type of ubiquitin chain as well as the molecules they are subset of signal transducers. Downstream effectors (e.g. the conjugated onto are likely to be stimulus specific. Interest- promoters of potential target genes) may integrate the combi- ingly, TNF and IL1b signaling appear to have differential nations of signal transducers (e.g. transcription factors) to requirements for ubiquitin chain types (161). While for determine the stimulus-specific activity level (e.g. of target IL-1b-induced IKK activation the Uev1A ⁄ Ubc13 ⁄ TRAF2 E3 genes). ligase complex that is restricted to K63 chain linkage is neces- Because they lack intrinsic enzymatic activities, TNFRs asso- sary for IKK activation, TNF-induced signaling does not ciate with TRAF proteins to initiate intracellular signaling. require catalysis of K63 chains (161). Recently, mass spec TRAF protein recruitment forms the basis for the encoding of analysis revealed that RIPK1 and NEMO are heavily ubiquiti- a combinatorial code by which a receptor-associated complex nated with K63, linear and K11-linked chains upon stimula- leads to the activation of several pleiotropic kinases. The fam- tion with TNF, indicating the high level of diversity in ily of TRAF proteins consists of 6 members, TRAF1-6, that ubiquitin modifications that occur during signaling (54). have both overlapping as well as distinct roles. The TRAF pro- More work is required to get a better understanding of the teins with which the receptor associates at least in part deter- stimulus specific role of ubiquitin chains. The use of ubiqu- mines the pathways that are activated. TRAFs are not only itin-chains as scaffolds allows for highly flexible and dynamic critically involved in signaling of TNFR superfamily members assembly of diverse signaling complexes but also for the but also play important roles in TLR ⁄ IL-1R signaling, where recruitment of specific activators and inactivators. Binding of TRAF3 is indispensable for IRF3 activation upon TLR3 engage- the scaffolds TAB 2 ⁄ 3 to polyubiquitin chains for example ment (157), or TRAF6 is necessary for activation of NFjBby recruits the MAP3K TAK1 to the TNFR1 complex to allow for LPS (101, 158). TRAFs have a RING-finger domain (RING) IKK2 activation, which in turn also depends on the ability of with an associated E3 ligase activity, a Zinc-finger (ZF) motif, its scaffold NEMO to bind to ubiquitin chains (39, 50–52, 55, and a highly conserved C-terminal domain that mediates 59). In addition to coordinating a signaling cascade, one can homo- and heterodimerization of TRAFs as well as association imagine that ubiquitin chains may branch one upstream signal with cell surface receptors (85, 159). to multiple downstream kinases through association of dis- The association of TRAF2 to CD40 is important for activa- tinct ubiquitin binding proteins to different types of poly- tion of MAPKs and to a lesser extent for the activation of ubiquitin chains, thereby playing a key role in encoding a canonical IKKs (67, 68, 86). Accordingly, TRAF2 deficiency combinatorial signaling code.

2011 John Wiley & Sons A/S • Immunological Reviews 244/2011 37 Schro¨felbauer & Hoffmann Æ Signaling speciﬁcity of TNFRSFs

The decoding of combinatorially coded signals has been Evidence for the temporal coding model for generating studied primarily at the level of gene expression. Gene pro- stimulus-specific signaling can be found in JNK, p38, and ERK moters or enhancers often contain binding sites for several signaling (170, 172). A prominent example demonstrating signal-responsive transcription factors that are the down- the importance of temporal control in MAPK signaling is the stream effectors of pleiotropic kinases. Synergistic function neuronal cell line PC12, in which ERK signaling can induce by several transcription factors has been documented in syn- proliferation (in response to EGF), as well as differentiation thetic experimental systems (162). The multivalent interac- (in response to NGF) depending on the duration of the signal tions by the ubiquitous co-activators CBP ⁄ p300 suggest a (173). While EGF induced MAPK signaling is transient, MAPK possible decoding mechanism in vivo. For some specific activity induced by NGF is sustained. Extensive evidence for genes, a combinatorial requirement has been established and the importance of temporal coding also comes from canonical examined at a mechanistic level. The classic example is the NFjB signaling. Quantitative measurements of the canonical control of the IFNb enhancer, which requires the activity of IKK activity revealed that its induced activity profile is stimu- AP-1, IFN response factor 3 (IRF3), and NFjB. A nucleo- lus-specific (171). Whereas TNF activates a transient IKK some located over the transcription start site was identified activity, LPS leads to prolonged IKK activity. In the former as the block for transcription initiation, and only the assem- case, the negative feedback regulator A20 limits late IKK activ- bly of an ‘enhanceosome’ (163) allowed for the recruitment ity, whereas in the latter, cytokine feedback via TNF ensures of chromatin remodeling factors that in turn allow for pre- an elevated late phase. Within the TNFRSF network, temporal initiation complex assembly (164). Interestingly, although control of effectors is mediated by the kinetics of key reaction AP-1 and NFjB are separated by four IRFs on the enhancer, mechanisms, such as receptor internalization, recycling, and protein-protein interactions between them were found to be replenishment, ubiquitin chain formation rates and their deg- critical (165). These are facilitated by HMG proteins capable radation through deubiquitin enzymes (DUSPs) such as A20, of inducing DNA bends (166). Similarly, expression of the nuclear translocation rates, and negative feedback mechanism chemokine MCP1 ⁄ CCL2 requires communication between a impacting IKK or NFjB(Fig. 5). proximally bound SP-1 and a distally bound NFjB that How may target genes distinguish between different tem- suggests chromosome looping (167). A different mechanism poral profiles of NFjB activity? We may imagine a variety of of combinatorial control has been proposed for the TNF gene. Expression of TNF requires NFjB binding to its promoter but also ERK and p38 signals to control mRNA nuclear-cytoplasmic transport, mRNA stability, and transla- tion (114, 168, 169). These examples indicate different mechanisms in the gene regulatory network may be targeted by the combinatorial control of TNFRSF-induced intracellular signals.

Temporal coding Biochemical cell population timecourse studies and single cell studies with in vivo reporters have revealed that the activity profile of TNFRS-induced transcription factors is dynamic and that the observed temporal profiles are stimulus-specific (170, 171). These observations led to the proposal that stimulus- Fig. 5. Temporal control can facilitate stimulus specificity. The tem- specific temporal control of pleiotropic transducers may allow poral profile of key signal transducers, whether pleiotropic kinases or for stimulus-specific signaling (7). The temporal code may be transcription factors, may determine signaling specificity. Temporal profiles are encoded and transduced through kinetic mechanisms that control encoded via receptor-associated mechanisms, transmitted via receptor internalization and recycling, half-life control of signaling medi- stimulus-specific temporal control of a pleiotropic transducer, ators, including ubiquitin chain second messengers or scaffolds, as well and decoded by mechanisms associated with the effector, for as negative and positive feedback. Signaling dynamics may be decoded to determine the activity of effectors, such as downstream genes, by mRNA example the gene regulatory network controlling the expres- half-life control, cooperativity with other transcription factors or chroma- sion of a target gene. tin regulatory mechanisms.

38 2011 John Wiley & Sons A/S • Immunological Reviews 244/2011 Schro¨felbauer & Hoffmann Æ Signaling speciﬁcity of TNFRSFs

mechanisms by which the temporal code may be decoded by begin to explain the mechanisms by which signaling specific- gene regulatory networks (GRNs). At its simplest, short versus ity is achieved. The most prominent of these may be the utili- long mRNA half-life may sensitize a gene to transient versus zation of scaffolding proteins, which may recruit kinases to prolonged NFjB activity. Coupled to non-linear or threshol- upstream activators or to downstream substrates. Indeed when ded dose response curves of promoters, the GRN may decode such scaffolds are multivalent, the same protein may deter- complex temporal profiles of NFjB in specific ways. In more mine both upstream activators and downstream targets, thus sophisticated GRNs, NFjB may need to coincident with or placing the kinase within a cascade and effectively insulating it phase shifted relative to other transcription factors to allow for from other signaling pathways. Within the MAPK field, prom- gene activation. On others yet, NFjB may be required to be inent examples of such scaffolds are the yeast Ste5 ⁄ Pbs2 scaf- coincident with a transcription factor induced by the early fold modules, and in mammals, the MAPK kinase cascades NFjB activity, forming a feed forward control loop. that are organized by various stimulus specific scaffolds. In NFjB signaling, NEMO appears to play a similar role (Schro¨- Conclusion felbauer et al. unpublished data). However, we expect that The signaling network innervated by members of the TNFR ongoing efforts to understand specificity of signaling within superfamily consists of a number of kinases with highly pleio- the TNFRSF network will lead to the identification of other tropic functions. Generally, these kinases show too little such scaffolding proteins and the characterization of their intrinsic specificity in vitro to account for their apparent signal- roles in signaling insulation and as mediators of signaling ing specificity in cells, but several models have emerged that crosstalk.

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