Commentary

AIP1: a new player in TNF signaling

M. Eugenia Guicciardi and Gregory J. Gores

Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota, USA

Address correspondence to: Gregory J. Gores, Mayo Medical School, Clinic, and Foundation, 200 First Street SW, Rochester, Minnesota 55905, USA. Phone: (507) 284-0686; Fax: (507) 284-0762; E-mail: gores.gregory@mayo.edu.

The signaling system generated by the binding of TNF to its cognate TNF-receptor (TNF-R) can contain two distinct type I transmembrane receptors, TNF-R1 and TNF-R2, and three ligands, the membrane-bound TNF-α, the soluble TNF-α, and the soluble lymphocyte-derived cytokine lymphotoxin-α (LT-α) (1). Both receptors are ubiquitously expressed in cells and interact with both forms of TNF-α, as well as with LT-α. However, TNF-R1 is the most potent in inducing cytotoxic signals. TNF-R1 is a typical death receptor with a 60- to 80-amino acid cytoplasmic sequence known as the death domain. The death domain, which is missing from TNF-R2, typically enables death receptors to initiate cytotoxic signals. Because of the role of TNF-α in human diseases (2), TNF-R1 signaling mechanisms are of considerable biomedical interest.

TNF-R1 signaling pathways

The intracellular signals originating from TNF-R1 are extremely complex and can lead to multiple, even opposite, cell responses including cell proliferation, inflammation, or cell death. Survival signals likely prevail over death signals under normal circumstances, given the resistance of most cells to TNF-α–induced toxicity. However, these cells can often be sensitized to TNF-α–mediated apoptosis by blocking protein or RNA synthesis, which suggests that neo-synthesis of protein is required to suppress the apoptotic stimulus. The expression of these antiapoptotic proteins is largely controlled by the activity of the transcription factor NF-κB, as inhibition of NF-κB also sensitizes cells to TNF-α–induced cell death (3).

Binding of TNF-R1 to TNF-α results in conformational changes in the receptor’s intracellular domain, resulting in rapid recruitment of several cytoplasmic death domain–containing adapter proteins via homophilic interaction with the death domain of the receptor (1). The first adaptor recruited to the clustered receptor is the TNF-R–associated protein with death domain, which functions as a docking protein for several signaling molecules, such as Fas-associated protein with death domain (FADD), TNF-R–associated factor-2 (TRAF-2), and receptor-interacting protein (RIP). Recruitment of FADD to the receptor promotes apoptosis by activation of caspase-8 and, possibly, caspase-10 (4). RIP associates with TRAF-2 to generate two distinct pathways (5). The first one signals through the activation of NF-κB–inducing kinase, NIK, and the catalytic IκB kinase complex, leading to phosphorylation of the NF-κB inhibitory protein IκB-α and translocation of NF-κB to the nucleus. The second pathway involves the MAPK cascade, and culminates in the activation of JNK/stress-activated protein kinase and p38 kinase. Both JNK and p38 play an important role in inflammatory responses. JNK has been described to phosphorylate and activate a number of transcription factors, including c-Jun, activating–transcription factor 2 and activator protein-1 (AP-1), which drive the expression of many proinflammatory molecules, including E-selectin, RANTES, IL-12, IL-6, and IL-8 (6). Activation of p38 is essential for production of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and for induction and expression of inflammatory-related enzymes such as COX-2 and iNOS (7).

MAPKs are activated by a well-recognized mechanism of dual phosphorylation mediated by one of the MAP kinase kinases (MAPKKs), which, in turn, are activated through phosphorylation by MAP kinase kinase kinases (MAPKKKs) (Figure 1a). Previous studies have shown that TNF-α–induced activation of JNK and p38 is mediated by the recruitment of the adaptor TRAF-2 and subsequent activation of the MAPKKK apoptosis signal–regulating kinase 1 (ASK1) (8). ASK1, a 170-kDa Ser/Thr kinase, activates the JNK and p38 MAPK signaling cascades in response to proinflammatory cytokines and oxidative stress. Overexpression of ASK1 in epithelial cells induces apoptosis, whereas a kinase-inactive mutant of ASK1 protects the cells from TNF-induced apoptosis, suggesting that ASK1 is involved in the TNF apoptotic signaling pathway (9). Functionally, ASK1 is composed of an inhibitory NH₂-terminal domain, an internal kinase domain, and a COOH-terminal regulatory domain. The COOH-terminal domain of ASK1 binds to the TRAF domain of TRAF-2 and TRAF-6 (8). The kinase domain is required for cytokine-induced JNK activation and cell death (9, 10). The NH₂-terminus contains an inhibitory domain that interacts with several cellular proteins and prevents ASK1 activation (11, 12). Although 14-3-3, a family of dimeric phosphoserine-binding molecules, binds to ASK1 specifically via Ser-967 in the COOH-terminal domain of ASK1, and inhibits ASK1-induced apoptosis (13), the precise role of 14-3-3 in regulating TNF-induced ASK1 activation has not been determined.

Figure 1

(a) Generic schema for the cytokine/adaptor/MAPK signaling paradigm. Cytokines, such as TNF-α, trigger oligomerization of their enzymatically inactive cognate receptors. The conformational change in the receptor complex following ligand binding facilitates recruitment of adaptor proteins. These adaptor proteins within the receptor complex interact directly or indirectly with upstream kinases (MAPKKK) permitting their activation with subsequent phosphorylation and activation of downstream kinases (MAPKK/MAPK). (b) Model of TNF-α signaling pathway through TRAF2/ASK1 employing the cytokine/adaptor/MAPK paradigm. Triggering of TNF-R1 leads to recruitment of the adaptor TRAF2, and concomitant activation of an unknown phosphatase via a mechanism yet to be identified. TRAF2 mediates the association of ASK1 with the newly identified Ras-GAP AIP1, which facilitates the release of ASK1 from its endogenous inhibitor 14-3-3. Disruption of the ASK1/14-3-3 complex and de-phosphorylation of ASK1 by the unknown phosphatase result in the activation of ASK1. ASK1, in turn, activates JNK via an MKK4- or MKK6-dependent pathway. Activation of JNK positively regulates the apoptotic machinery by transcription-dependent (i.e., activation of the transcription factor AP-1) and transcription-independent (i.e., phosphorylation and activation of proapototic Bcl-2 proteins Bim and Bmf) mechanisms. P, phosphate.

Regulation of ASK-1

In this issue of the JCI, Zhang and coworkers have described a new Ras-GTPase–activating protein (Ras-GAP), ASK1-interacting protein-1 (AIP1), which promotes TNF-induced activation of ASK1 by facilitating the dissociation of ASK1 from its endogenous inhibitor 14-3-3 (14). The authors demonstrate that TNF-α activates AIP1 by causing AIP1 to unfold and expose its NH₂-terminal domain, rendering it available for interacting with the COOH-terminal domain of ASK1. The binding of AIP1 to ASK1 then disrupts the ASK1/14-3-3 complex (Figure 1b). Since the dissociation of ASK1 from 14-3-3 has been previously described to be accompanied by de-phosphorylation of ASK1 at Ser-967 by an unknown phosphatase (15), the authors hypothesize that AIP1 may act as a chaperone, recruiting the phosphatase to ASK1. Supporting this interpretation, their data show that the binding site of AIP1 on ASK1 lies in a sequence surrounding the 14-3-3 binding site, and that AIP1, unlike 14-3-3, has higher affinity for the de-phosphorylated form of ASK1 at Ser-967. However, the nature of this unknown phosphatase and the temporal sequence of the events leading to the dissociation of the ASK1/14-3-3 complex remain to be elucidated. In fact, the identification, localization, and function of this phosphatase will be necessary to confirm and establish the proposed model. If proved, this model of ASK1 de-phosphorylation as an activation step for a MAPKKK would be unique.

The present paper from Zhang and coworkers (14) represents a significant scientific contribution by identifying the missing direct target of TRAF-2 in the JNK pathway, AIP1. Regulation of ASK1 by AIP1 is likely very important in TNF-α–mediated cytotoxic signaling as ASK1 is upstream of JNK. Activation of JNK can lead to apoptosis by transcriptionally-dependent and independent processes. JNK activation of the transcription factor AP-1 has been implicated in various models of cytotoxicity (16). Moreover, in a recent study, Lei and Davis describe a transcriptionally-independent mechanism for JNK associated apoptosis by demonstrating that JNK directly phosphorylates the proapoptotic BH3-only proteins Bim and Bmf promoting mitochondrial dysfunction with cytochrome c release (17). The role of ASK1 in initiating this cascade requires further clarification.

This paper also highlights the role of 14-3-3 in regulating apoptosis. Phosphorylation of Bad, another proapoptotic BH3-only member of the Bcl-2 family, also promotes its recruitment and sequestration by 14-3-3 proteins (18, 19). This mechanism of inactivation of Bad is highly reminiscent of the one operated by 14-3-3 on ASK1, suggesting that one of the functions of 14-3-3 proteins is to support cell survival by inhibiting the activity of proapoptotic proteins.

In conclusion, the identification of AIP1 by Zhang and co-workers (14) provides a further detail of the TNF-R1 MAPK cascade and helps to unravel the molecular bases of the TNF signaling. Given its important contribution to TNF-induced JNK activation, AIP1 may represent a suitable target for possible therapeutic applications in human diseases characterized by increased TNF-α–mediated apoptosis.

Acknowledgments

This work was supported by grants from the NIH (DK 63947) and the Mayo Foundation.

Footnotes

See the related article beginning on page 1933.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: TNF receptor (TNF-R); lymphotoxin-α (LT-α); Fas-associated protein with death domain (FADD); TNF receptor–associated factor-2 (TRAF-2); receptor-interacting protein (RIP); apoptosis signal–regulating kinase 1 (ASK1); ASK1-interacting protein (AIP1); MAPK kinase (MAPKK); MAPK kinase kinase (MAPKKK); Ras GTPase-activating protein (Ras-GAP).

References

1. Wallach, D, et al. Tumor necrosis factor receptor and Fas signaling mechanisms. Ann. Rev. Immunol. 1999. 17:331-367.
   View this article via: PubMed CrossRef
2. McDermott, MF. TNF and TNFR biology in health and disease. Cell. Mol. Biol. 2001. 47:619-635.
   View this article via: PubMed
3. Wang, CY, Mayo, MW, Korneluk, RG, Goeddel, DV, Baldwin, AS. NF-κB antiapoptosis: induction of TRAF 1 and TRAF 2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998. 281:1680-1683.
   View this article via: PubMed CrossRef
4. Kischkel, FC, et al. Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8. J. Biol. Chem. 2001. 276:46639-46646.
   View this article via: PubMed CrossRef
5. Wajant, H, Scheurich, P. Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling. Int. J. Biochem. Cell. Biol. 2001. 33:19-32.
   View this article via: PubMed CrossRef
6. Ip, YT, Davis, RJ. Signal transduction by the c-jun N-terminal kinase (JNK): from inflammation to development. Curr. Opin. Cell Biol. 1998. 10:205-219.
   View this article via: PubMed CrossRef
7. Ono, K, Han, J. The p38 signal transduction pathway: activation and function. Cell Signal. 2000. 12:1-13.
   View this article via: PubMed CrossRef
8. Nishitoh, H, et al. ASK1 is essential for JNK/SAPK activation by TRAF2. Mol. Cell. 1998. 2:389-395.
   View this article via: PubMed CrossRef
9. Chang, HY, Nishitoh, H, Yang, X, Ichijo, H, Baltimore, D. Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx. Science. 1998. 281:1860-1863.
   View this article via: PubMed CrossRef
10. Ichijo, H, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science. 1997. 275:90-94.
    View this article via: PubMed CrossRef
11. Liu, H, Nishitoh, H, Ichijo, H, Kyriakis, JM. Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin. Mol. Cell. Biol. 2000. 20:2198-2208.
    View this article via: PubMed CrossRef
12. Saitoh, M, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. EMBO J. 1998. 17:2596-2606.
    View this article via: PubMed CrossRef
13. Zhang, L, Chen, J, Fu, H. Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins. Proc. Natl. Acad. Sci. U. S. A. 1999. 96:8511-8515.
    View this article via: PubMed CrossRef
14. Zhang, R, et al. AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. J. Clin. Invest. 2003. 111:1933-1943. doi:10.1172/JCI200317790.
    View this article via: JCI.org PubMed
15. Liu, Y, Yin, G, Surapisitchat, J, Berk, BC, Min, W. Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3. J. Clin. Invest. 2001. 107:917-923.
    View this article via: JCI.org PubMed CrossRef
16. Jones, BE, Czaja, MJ. Mechanisms of hepatic toxicity. III. Intracellular signaling in response to toxic liver injury. Am. J. Physiol. Gastrointest. Liver Physiol. 1998. 275:G874-G878.
17. Lei, K, Davis, RJ. JNK phosphorylation of Bim-related members of the Bcl-2 family induces Bax-dependent apoptosis. Proc. Natl. Acad. Sci. U. S. A. 2003. 100:2432-2437.
    View this article via: PubMed CrossRef
18. Zha, J, Harada, H, Yang, E, Jockel, J, Korsmeyer, SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not Bcl-X_L. Cell. 1996. 87:619-628.
    View this article via: PubMed CrossRef
19. Datta, SR, et al. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol. Cell. 2000. 6:41-51.
    View this article via: PubMed CrossRef