IκB kinase-induced interaction of TPL-2 kinase with PNAS PLUS 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases Abduelhakem Ben-Addia,1, Agnes Mambole-Demaa,1, Christine Brendera, Stephen R. Martinb, Julia Janzena, Sven Kjaerc, Stephen J. Smerdond, and Steven C. Leya,2 Divisions of aImmune Cell Biology, bPhysical Biochemistry, dMolecular Structure, Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom; and cProtein Purification Facility (PPF), Cancer Research UK, London Research Institute, London WC2A 3PX, United Kingdom Edited by Philip Cohen, University of Dundee, Dundee, United Kingdom, and approved April 10, 2014 (received for review November 1, 2013) The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activa- In unstimulated cells, TPL-2 forms a stoichiometric complex tion of the ERK-1/2 MAP kinase pathway during inflammatory with NF-κB1 p105, an NF-κB inhibitory protein and the precursor responses, but it can transform cells following C-terminal truncation. of the NF-κB p50 subunit (12, 13), and A20-binding inhibitor of IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus NF-κB (ABIN)-2 (14). Interactions with both p105 and ABIN-2 regulates full-length TPL-2 activation of ERK-1/2 by a mechanism are required to maintain TPL-2 protein stability (12, 15, 16). that has remained obscure. Here, we show that TPL-2 Ser-400 phos- Binding to p105 also prevents access of TPL-2 to its substrates phorylation by IKK and TPL-2 Ser-443 autophosphorylation cooper- MEK-1/2. Consequently, LPS activation of TPL-2 MEK-1/2 ki- ated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 nase activity requires the release of TPL-2 from p105 (16, 17). to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase This event is triggered by phosphorylation of p105 by the IκB activity, which was essential for TPL-2 activation of ERK-1/2. The kinase (IKK) complex, which induces p105 K48-linked ubiquiti- binding of 14-3-3 to TPL-2 was also indispensible for lipopolysac- nation and subsequent proteolysis by the proteasome (17, 18). charide-induced production of tumor necrosis factor by macro- Although NF-κB1 p105 interaction prevents phosphorylation phages, which is regulated by TPL-2 independently of ERK-1/2 of MEK-1/2 by TPL-2, it does not inhibit TPL-2 catalytic activity IMMUNOLOGY activation. Our data identify a key step in the activation of TPL-2 (19, 20). LPS stimulation is still required for activation of ERK- − − signaling and provide a mechanistic insight into how C-terminal 1/2 by TPL-2 ectopically expressed in Nfkb1 / macrophages, deletion triggers the oncogenic potential of TPL-2 by rendering its which express virtually undetectable amounts of endogenous kinase activity independent of 14-3-3 binding. TPL-2 (20). The signaling activity of TPL-2 is therefore regu- lated independently of its release from p105. This regulation inflammation | NF-κB | MAP3K8 involves the inducible phosphorylation of TPL-2 on serine 400 (S400) in its C-terminal tail (20). Mutation of this conserved ells of the innate immune system are rapidly activated fol- residue to alanine blocks the ability of ectopically expressed Clowing infection via stimulation of germ-line–encoded pattern TPL-2 to induce activation of ERK-1/2 in LPS-stimulated − − recognition receptors (PRRs) that detect pathogen-associated Nfkb1 / macrophages. Thus, TPL-2 activation of the ERK-1/2 molecular patterns (PAMPs) (1). The Toll-like receptor (TLR) MAP kinase pathway requires TPL-2 phosphorylation on S400. family of PRRs recognizes a wide range of PAMPs, including We have recently demonstrated that TPL-2 S400 phosphor- lipids, lipoproteins, proteins, and nucleic acids from bacteria, viruses, ylation is mediated by IKK2, a catalytic subunit of the IKK parasites, and fungi (2). TLRs trigger signaling pathways, leading to the activation of nuclear factor kappa light-chain enhancer of acti- Significance vated B cells (NF-κB) transcription factors, IFN-regulatory factors, and each of the major mitogen-activated protein (MAP) kinase TPL-2 is a MEK-1/2 kinase that mediates Toll-like receptor ac- subtypes [extracellular signal-regulated kinases 1 and 2 (ERK-1/2), tivation of ERK-1/2 MAP kinases in macrophages and is critical c-Jun N-terminal kinases, and p38α/β]. Together, these signal- for TNF induction during inflammation. TPL-2 activation of ing processes induce the expression of hundreds of proteins, MEK-1/2 requires release from its associated inhibitor NF-κB1 which regulate multiple aspects of the innate immune response. p105, resulting from p105 proteolysis triggered by the IκB ki- Activation of ERK-1/2 MAP kinases in macrophages by all nase (IKK) complex. Here, we show that IKK phosphorylation TLRs is mediated by the MAP 3-kinase TPL-2 (tumor progression of the TPL-2 C terminus induces 14-3-3 association with TPL-2, locus-2, also known as Cot and MAP3K8), which phosphorylates stimulating its MEK kinase activity, which is essential for TPL-2 and activates the ERK-1/2 kinases, mitogen-activated ERK kinase- activation of ERK-1/2. The 14-3-3 binding to TPL-2 is also 1 and -2 (MEK-1 and -2) (3, 4). TPL-2 regulates cytokine pro- indispensible for its induction of TNF, which is regulated indepen- duction by TLR-stimulated macrophages and dendritic cells, dently of ERK-1/2 activation. The IKK complex, a key regulator of inducing production of tumor necrosis factor (TNF), interleukin NF-κB transcription factors, therefore directly controls two key (IL)-1β, and IL-10, while suppressing production of IL-12 and steps for TPL-2 activation in inflammatory responses. IFN-β (5–8). Although TPL-2 has complex effects on the pro- duction of pro- and anti-inflammatory cytokines by myeloid cells, Author contributions: A.B.-A., A.M.-D., S.R.M., S.J.S., and S.C.L. designed research; A.B.-A., −/− A.M.-D., S.R.M., and J.J. performed research; C.B., J.J., and S.K. contributed new reagents/ experiments with Map3k8 mice indicate that the net effect analytic tools; A.B.-A., A.M.-D., S.R.M., and S.C.L. analyzed data; and S.C.L. wrote of TPL-2 signaling in the innate immune system is to induce the paper. inflammation (3). For example, TPL-2 signaling promotes TNF- The authors declare no conflict of interest. induced endotoxin shock (5), is required for the development of This article is a PNAS Direct Submission. TNF-induced inflammatory bowel disease (9), and regulates the 1A.B.-A. and A.M.-D. contributed equally to this work. onset and severity of experimental autoimmune encephalomyelitis, 2To whom correspondence should be addressed. E-mail: [email protected]. a model for multiple sclerosis (10). TPL-2 is consequently consid- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ered a potential drug target in certain autoimmune diseases (11). 1073/pnas.1320440111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1320440111 PNAS Early Edition | 1of10 Downloaded by guest on October 2, 2021 complex (21). However, it remained unclear how IKK2 phos- unaffected by Map3k8S400A mutation (Fig. S2F). Map3k8S400A phorylation of this residue controls TPL-2 signaling. In the pres- mutation also fractionally reduced p38α phosphorylation after ent study, we show that S400 phosphorylation was critical for the stimulation with TNF and CpG, consistent with earlier results association of 14-3-3 with the TPL-2 C terminus. Binding of 14-3- suggesting that p38α activation by these stimuli was mediated in 3 increased the efficiency of MEK-1 phosphorylation by TPL-2 in part by the NF-κB1 p105/TPL-2 pathway (22). Together, these vitro and was essential for TPL-2 activation of the ERK-1/2 MAP data provided unequivocal genetic evidence that S400 is essential kinase pathway in cells. Thus, the IKK complex, the principal for TPL-2–dependent activation of ERK-1/2 in macrophages. regulator of NF-κB transcription factors, directly controls both of TPL-2 promotes the production of soluble TNF (sTNF) in- the key steps required for TPL-2 to phosphorylate MEK-1/2 and dependently of IKK-induced release of TPL-2 from p105 and activate the ERK-1/2 MAP kinase pathway in inflammation. its ability to activate ERK-1/2 (22). Map3k8S400A/S400A BMDMs produced very low levels of sTNF after LPS stimulation, which Results was reduced by ∼90% compared with WT BMDMs (Fig. 1C). S400 Is Essential for TPL-2 Signaling in Macrophages. We previously Furthermore, production of sTNF in vivo after i.p. LPS injection investigated the role of S400 in LPS activation of TPL-2 sig- was substantially reduced in Map3k8S400A/S400A compared with naling in macrophages by retroviral overexpression of TPL-2 in WT controls (Fig. 1D). These results imply that Map3k8S400A − − Map3k8 / macrophages (20). However, the molecular mecha- mutation blocked TPL-2 phosphorylation of the unknown target nism by which S400 phosphorylation regulated TPL-2 signaling protein(s) by which TPL-2 controls sTNF production. remained unclear. To address this question, we first determined whether S400 was required for signaling by TPL-2 under physi- TPL-2 S400 Does Not Regulate TPL-2 Release from NF-κB1 p105. LPS ological conditions by generating the Map3k8S400A/S400A mouse activation of ERK-1/2 requires TPL-2 release from p105 to fa- strain, which expressed mutant TPL-2S400A (Fig. S1). Immunoblot cilitate MEK-1/2 phosphorylation (17).