IκB -induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP

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 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 -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.

E2394–E2403 | PNAS | Published online May 27, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1320440111 Downloaded by guest on September 30, 2021 complex (21). However, it remained unclear how IKK2 phos- unaffected by Map3k8S400A mutation (Fig. S2F). Map3k8S400A PNAS PLUS 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). To investigate whether analysis of bone marrow-derived macrophages (BMDMs) gen- Map3k8S400A mutation affected this step in TPL-2 activation, erated from these mice demonstrated that the Map3k8S400A muta- BMDM lysates were depleted of p105 and then immunoblotted tion did not affect steady-state levels of TPL-2, but completely for TPL-2. No p105-free TPL-2 was detected in unstimulated WT blocked TPL-2–dependent activation of ERK-1/2 following LPS or Map3k8S400A/S400A cells (Fig. 2A). After 7.5 min of LPS stimu- stimulation (Fig. 1A).LPSactivationofp38α was fractionally lation, similar amounts of M1–TPL-2 and M30–TPL-2 [pro- reduced in Map3k8S400A/S400A macrophages compared with WT, duced by alternative translational initiation on a second me- − − as previously found with LPS-stimulated Map3k8 / macrophages thionine at residue 30 (25)] were detected in the p105-depleted (7). To investigate the effect of S400A mutation on ERK-1/2 WT and Map3k8S400A/S400A lysates. These results demonstrated activation in vivo, mice were injected intraperitoneally (i.p.) with that Map3k8S400A mutation did not block TPL-2 activation of LPS (22). Intracellular staining clearly detected phospho–ERK-1/2 ERK-1/2 by preventing TPL-2 release from its inhibitor p105. + in WT peritoneal F480 macrophages 10 min after LPS injection We next determined whether Map3k8S400A mutation inhibited (Fig. 1B). In contrast, no phospho–ERK-1/2 signal was detected the catalytic activity of TPL-2 by in vitro kinase assay. In WT − − in Map3k8S400A/S400A macrophages, similar to Map3k8 / macro- BMDMs, LPS stimulation induced a marked increase in TPL-2 phages (22), demonstrating that S400 was essential for TPL-2– MEK-1 kinase activity (Fig. 2B). LPS induced the MEK-1 kinase dependent activation of ERK-1/2 in vivo. activity of TPL-2S400A to a similar degree, although phosphory- The Map3k8S400A mutation additionally blocked induction of lation of endogenous MEK-1/2 was completely blocked in LPS- ERK-1/2 phosphorylation in BMDMs by TNF, CpG (TLR9), stimulated Map3k8S400A/S400A cells. These results showed that Pam-3-Cys-4 (TLR2), poly-(I:C) (TLR3), flagellin (TLR5), and S400 is not required for TPL-2 catalytic activity in vitro, but is imiquimod (TLR7) (Fig. S2 A–E), which all induce ERK-1/2 essential to couple TPL-2 to downstream signaling pathways in activation via TPL-2 (7, 8, 22–24). However, activation of ERK- cells, consistent with an earlier study analyzing transfected TPL-2 1/2 by phorbol ester, which does not require TPL-2 (5), was in Jurkat T cells (26). IMMUNOLOGY

Fig. 1. Map3k8S400A mutation blocks LPS activation of ERK-1/2 in macrophages. (A) WT and Map3k8S400A/S400A BMDMs were stimulated with LPS for the indicated times. Lysates were immunoblotted. (B) WT and Map3k8S400A/S400A mice were injected intraperitoneally (i.p.) with LPS or PBS control. After 10 min, peritoneal cells were aspirated and stained for surface F4/80 and intracellular phospho–ERK-1/2 (P-ERK). Anti–P-ERK mean fluorescence intensity (MFI) of F4/ + 80 cells was determined by flow cytometry (mean ± SEM; n = 6 mice per genotype). (C) Triplicate cultures of BMDMs were stimulated with LPS for the indicated times, and TNF levels in supernatants were assayed. (D) WT and Map3k8S400A/S400A mice were injected i.p. with LPS. After 1 h, mice were bled, and serum TNF levels were quantified (mean ± SEM; n = 8 mice per genotype). **P < 0.01; ***P < 0.001; ****P < 0.0001.

Ben-Addi et al. PNAS | Published online May 27, 2014 | E2395 Downloaded by guest on September 30, 2021 Fig. 2. Map3k8S400A mutation does not affect LPS-induced release of TPL-2 from NF-κB1 p105 or TPL-2 intrinsic kinase activity. WT and Map3k8S400A/S400A (Map3k8S400A) BMDMs were stimulated with LPS. (A) Total and p105-depleted lysates were immunoblotted. (B) TPL-2 was immunoprecipitated and assayed for its ability to phosphorylate GST–MEK-1K207A in vitro. MEK-1 phosphorylation and TPL-2 levels were assayed by immunoblotting.

S400 Phosphorylation Is Required for TPL-2 Interaction with 14-3-3. peptides containing consensus Raf 14-3-3 binding sites (28). Fur- Because S400 was not required for TPL-2 intrinsic catalytic activity thermore, pulldown experiments with GST–14-3-3γ demonstrated or release from p105, we hypothesized that S400 phosphoryla- that TPL-2 could interact with 14-3-3 after LPS stimulation of WT tion might promote TPL-2 interaction with a phospho-peptide primary macrophages (Fig. 3B). However, TPL-2S400A from LPS- binding protein. Scansite (27) was used to determine whether stimulated Map3k8S400A/S400A cells did not bind to GST–14-3-3γ. TPL-2 phospho-S400 corresponded to any known binding sites. LPS stimulation of WT macrophages also induced interaction of This analysis revealed that the sequence around phospho-S400 TPL-2 with GST–14-3-3e,GST–14-3-3ζ, and GST–14-3-3η (Fig. (RCQpSLD, where pS represents phospho-serine) had similarity S3A). Similarly, Myc–TPL-2 transiently expressed in IL-1R–293 to a mode I 14-3-3 (RSXpSXP) at low stringency cells interacted with GST–14-3-3γ after IL-1β stimulation, whereas (28). Consistent with this prediction, biolayer interferometry dem- Myc–TPL-2S400A did not (Fig. S3B). Furthermore, Flag–TPL-2 – onstrated that a synthetic TPL-2393 407 phosphopeptide corre- immunoprecipitated from transiently transfected IL-1R–293 cells sponding to the sequence surrounding S400 bound to recombinant associated with endogenous 14-3-3 after IL-1β stimulation, and 14-3-3ζ with a Kd of 0.7 μM, whereas the corresponding non- this interaction was blocked by S400A mutation (Fig. 3C). phosphorylated peptide did not bind (Fig. 3A). This affinity was Phosphorylation of TPL-2 S400 is mediated by IKK2 (21), similar to those reported for 14-3-3 interaction with phospho- and 14-3-3 dimers bind to their client proteins via phosphor-

– Fig. 3. S400 is required for TPL-2 interaction with 14-3-3. (A) Biolayer interferometry was used to measure the ability of TPL-2393 407 peptide non- phosphorylated (□) and phosphorylated on S400 (●) to displace 14-3-3ζ protein associated with biotinylated phospho-S621–cRaf peptide bound to

a Streptavidin sensor chip. A Kd value of 0.7 μM for the phospho-S400 peptide was calculated. (B)GST–14-3-3γ pulldowns from lysates of BMDMs with or without LPS (15 min) were immunoblotted. (C) IL-1R–293 cells were transiently transfected with expression constructs encoding WT or S400A Flag–TPL-2 or empty vector (EV). Anti-Flag immunoprecipitates and cell lysates were immunoblotted. (D) BMDMs were preincubated with BI605906 IKK2 inhibitor or vehicle control for 1 h, before stimulation with LPS for 15 min. GST–14-3-3γ pulldowns from cells lysates were immunoblotted. (E and F) BMDMs of the indicated genotypes were stimulated with LPS or left unstimulated. GST–14-3-3γ pulldowns and total cell lysates were immunoblotted.

E2396 | www.pnas.org/cgi/doi/10.1073/pnas.1320440111 Ben-Addi et al. Downloaded by guest on September 30, 2021 ylated serine/threonine motifs (29). In accordance with this ob- TPL-2 Interaction with 14-3-3 Regulates TPL-2 Activation of ERK-1/2. PNAS PLUS servation, pharmacological inhibition of IKK2 in macrophages We next investigated whether the S400A mutation abrogated blocked LPS-induced interaction of TPL-2 with GST–14-3-3γ TPL-2 activation of ERK-1/2 by preventing recruitment of 14-3-3 (Fig. 3D). IKK2 phosphorylation of S400, therefore, was re- dimers. To test this hypothesis, an expression construct was quired for TPL-2 binding to GST–14-3-3γ, demonstrating why generated in which the TPL-2 S400 14-3-3 binding site was Map3k8S400A mutation ablated TPL-2 interaction with 14-3-3 replaced with the R18 peptide motif that binds with high affinity in agonist-stimulated cells. to 14-3-3 proteins in a phosphorylation-independent fashion Analysis of Nfkb1SSAA/SSAA BMDMs, in which the IKK target (30). In vitro kinase assays demonstrated that introduction of the serines on NF-κB1 p105 are mutated to alanine to prevent signal- R18 peptide into the S400 binding site did not alter the basal catalytic activity of TPL-2 (Fig. S5A). The resulting chimeric induced p105 proteolysis, has demonstrated that TPL-2 catalytic – protein Myc–TPL-2S400 R18 was then transiently expressed in IL- activity induces the production of sTNF by macrophages in- – 1R–293 cells. As expected, Myc–TPL-2S400 R18 interacted constitu- dependently of its release from p105 and activation of ERK-1/2 – γ – (22). Furthermore, TPL-2 can be phosphorylated on S400 while tively with GST 14-3-3 , whereas WT Myc TPL-2 only bound S400A after IL-1β stimulation (Fig. 4A). IL-1β induced the phos- still associated with p105 (20). Because Map3k8 mutation SSAA/SSAA phorylation of endogenous MEK-1/2 in cells expressing Myc– inhibited LPS induction of sTNF by Nfkb1 BMDMs – TPL-2S400 R18,similartoWTMyc–TPL-2. In contrast, Myc– (Fig. S4A), in which LPS-induced release of TPL-2 from p105 – TPL-2S400 R18KK, in which two key negatively charged residues in is blocked (22), we speculated that TPL-2 could associate with the core binding motif of the R18 sequence were replaced with 14-3-3 while still complexed with p105. Consistent with this hy- – γ β Nfkb1SSAA/SSAA lysines, did not interact with GST 14-3-3 or facilitate IL-1 pothesis, LPS stimulation of BMDMs promoted activation of MEK-1/2, similar to Myc–TPL-2S400A (Fig. 4A and – γ – the interaction of GST 14-3-3 with TPL-2, although at much Fig. S3B). Binding of TPL-2S400 R18 to 14-3-3, therefore, replaced lower levels than with LPS-stimulated WT cells, and also p105 the requirement of S400 phosphorylation for TPL-2–mediated ac- E – γ (Fig. 3 ). TPL-2 was not detected in GST 14-3-3 pulldowns tivation of ERK-1/2 in IL-1R–293 cells. after immunodepletion of p105 from lysates of LPS-stimulated To confirm the physiological significance of these data, TPL- SSAA/SSAA – − − Nfkb1 BMDMs (Fig. S4B). Furthermore, GST–14-3-3γ 2S400 R18 was expressed in Map3k8 / BMDMs by retroviral S400A did not bind to TPL-2 in lysates of LPS-stimulated transduction. As shown previously (20), WT TPL-2 rescued LPS Nfkb1SSAA/SSAA Map3k8S400A/S400A BMDMs (Fig. 3F). There- induction of ERK-1/2 phosphorylation, whereas no ERK-1/2 fore, TPL-2 could interact inefficiently with 14-3-3 while bound phosphorylation was detected in cells expressing TPL-2S400A to p105, and this association was dependent on TPL-2 S400. (Fig. 4B). Importantly, LPS stimulation clearly induced ERK-1/2 – Together, these results indicate that IKK2 phosphorylation of phosphorylation in cells transduced with TPL-2S400 R18, whereas – S400 triggered binding of TPL-2–14-3-3, and this interaction expression of TPL-2S400 R18KK had no effect on LPS induction of could occur when TPL-2 was still associated with p105. ERK-1/2 phosphorylation. The level of ERK-1/2 phosphoryla- IMMUNOLOGY

Fig. 4. Reconstitution of TPL-2 signaling activity with 14-3-3–binding R18 motif. (A) IL-1R–293 cells, transiently transfected with empty vector (EV) or plasmids – encoding Myc-tagged TPL-2, TPL-2S400A, and TPL-2S400 R18, were stimulated with IL-1β or left unstimulated. (B–D)GST–14-3-3γ pulldowns and lysates were − − − − immunoblotted. BMDMs generated from Map3k8 / (B and C)orNfkb1 / (D) mice were transduced with retroviruses encoding the indicated TPL-2 proteins. In B and D, cells were stimulated with LPS for 15 min or left unstimulated, and lysates were immunoblotted. In C, cells were stimulated with LPS for 9 h, and sTNF in culture supernatants was assayed (mean ± SEM; n = 3). ****P < 0.0001.

Ben-Addi et al. PNAS | Published online May 27, 2014 | E2397 Downloaded by guest on September 30, 2021 − − tion was higher in LPS-stimulated Map3k8 / cells expressing (RYGpTVED), and S443 (R/KRQRpSLY) as potential phos- – WT TPL-2 than TPL-2S400 R18, which may reflect in part the pho-peptide binding sites for 14-3-3. Biolayer interferometry – relative TPL-2 and TPL-2S400 R18 protein expression levels. confirmed that synthetic TPL-2 phosphopeptides corresponding – Expression of TPL-2S400 R18 was also found to rescue LPS- to S62 and S443 both bound to 14-3-3ζ (Fig. 5A and Fig. S6A), − − induced sTNF production by Map3k8 / BMDMs similar to WT although with slightly lower affinities than that for TPL-2 S400 TPL-2, whereas TPL-2S400 did not (Fig. 4C). phosphopeptide. No binding was detected with the T80 TPL-2 Together, these results suggested that S400 was essential for phosphopeptide. These results raised the possibility that phos- TPL-2 activation of ERK-1/2 due to its ability to mediate re- pho-S62 or -S443 might function cooperatively with phospho- cruitment of 14-3-3 after IKK2-mediated phosphorylation. How- S400 to mediate TPL-2 interaction with 14-3-3 dimers. We had ever, although constitutively bound to 14-3-3, agonist stimulation previously shown that TPL-2 S62A mutation does not affect – was nevertheless required for TPL-2S400 R18 activation of ERK-1/2 TPL-2 activation of ERK-1/2 in macrophages (21). Furthermore, − − in both IL-1R–293 cells and Map3k8 / BMDMs. Thus, 14-3-3 TPL-2S62A, expressed in IL-1R–293 cells, was still able to interact binding to the TPL-2 C terminus per se was insufficient to promote with GST–14-3-3γ and mediate MEK-1/2 activation after IL-1β – TPL-2–dependent activation of ERK-1/2. Because TPL-2S400 R18 stimulation (Fig. S6B). Consequently, phosphorylation of S62 was still able to interact with p105 (Fig. S5B), it was possible that was not required for TPL-2–dependent activation of ERK-1/2, this observation simply reflected a requirement for stimulus-in- and further experiments focused on determining the role of S443 – duced release of TPL-2S400 R18 from p105. To investigate this in TPL-2 signaling. – − − possibility, TPL-2S400 R18 was expressed in Nfkb1 / BMDMs, Immunoblotting with a TPL-2 phospho-S443 antibody revealed − − which lack endogenous p105 expression. Similar to Map3k8 / that IL-1β stimulation induced the phosphorylation of S443 on – BMDMs, LPS stimulation was required for TPL-2S400 R18 to induce Myc–TPL-2 expressed in IL-1R–293 cells (Fig. 5B). Analysis of − − ERK-1/2 phosphorylation in Nfkb1 / BMDMs (Fig. 4D). There- Myc–TPL-2S400A and Myc–TPL-2S443A mutants demonstrated that fore, activation of ERK-1/2 was still dependent on agonist stimu- TPL-2 S400 and S443 could be phosphorylated independently of – lation when TPL-2S400 R18 was constitutively bound to 14-3-3 and one another. Pharmacological inhibition of TPL-2 catalytic activity free from p105-mediated inhibition. with C34 small-molecule inhibitor (32) blocked TPL-2 S443 phosphorylation, whereas phosphorylation on TPL-2 S443 was S443 Is Required for Optimal TPL-2 Interaction with 14-3-3. The 14-3-3 largely unaffected by inhibition of IKK2 with BI605906 (Fig. 5C). dimers contain two independent ligand-binding sites, and These data suggested that IL-1β stimulation induced the auto- consequently can interact simultaneously with two phospho- phosphorylation of TPL-2 on S443. Accordingly, IL-1β stimula- peptide-binding motifs, found either on single or separate cli- tion did not induce S443 phosphorylation of catalytically inactive ent proteins (31). In addition to S400, Scansite analysis of the TPL-2D270A (Fig. 5D). However, because it was not possible to TPL-2 amino acid sequence identified S62 (RSKpSLLL), T80 detect in vitro S443 phosphorylation by using affinity-purified

Fig. 5. The 14-3-3 interaction with phospho-S443 is required for optimal TPL-2 signaling. (A) Binding between 14-3-3ζ and a synthetic phospho-S443–TPL- 436–450 2 peptide as in Fig. 3A.AKd value of 1.9 ± 0.4 μM was calculated. (B) IL-1R–293 cells, transiently transfected with plasmids encoding WT, S400A, or S443 AMyc–TPL-2, were stimulated with IL-1β or left unstimulated. Anti-Myc immunoprecipitates and cell lysates were immunoblotted. (C) IL-1R–293 cells expressing Myc–TPL-2 were pretreated with C34 (TPL-2 inhibitor), BI605906 (IKK2 inhibitor), or vehicle control and then stimulated with IL-1β or left unsti- mulated. Anti-Myc immunoprecipitates were immunoblotted. (D) IL-1R–293 cells expressing Myc–TPL-2 or kinase-inactive Myc–TPL-2D270A were stimulated with IL-1β or left unstimulated. Anti-Myc immunoprecipitates were immunoblotted. (E) IL-1R–293 cells transiently expressing the indicated Myc–TPL-2 pro- − − teins were stimulated with IL-1β or left unstimulated. GST–14-3-3γ pulldowns and lysates were immunoblotted. (F) Map3k8 / BMDMs, transduced with retroviruses encoding the indicated TPL-2 proteins, were stimulated with LPS (15 min) or left unstimulated. Lysates were immunoblotted.

E2398 | www.pnas.org/cgi/doi/10.1073/pnas.1320440111 Ben-Addi et al. Downloaded by guest on September 30, 2021 TPL-2, it cannot formally be ruled out that TPL-2 S443 phos- introduction of the R18 motif into the S443 14-3-3 binding site PNAS PLUS phorylation was mediated by a downstream kinase activated by removed the need for S443 phosphorylation, but not agonist stim- – TPL-2 catalytic activity. ulation, for TPL-2 signaling. However, TPL-2S443 R18 did not S443A mutation reduced the interaction of Myc–TPL-2 with signal as efficiently as WT TPL-2 in either IL-1R–293 cells or − − GST–14-3-3γ induced by IL-1β stimulation (Fig. 5E), similar to Map3k8 / BMDMs. – – S400A mutation (Fig. 3C). Consistent with the requirement for Both TPL-2S400 R18 and TPL-2S443 R18 chimeric proteins were TPL-2 to interact with 14-3-3 to activate the ERK-1/2 MAP ki- able to constitutively bind to 14-3-3, but still required IL-1β nase pathway, IL-1β induction of MEK-1/2 phosphorylation was stimulation to mediate activation of MEK-1/2 in IL-1R–293 cells − − substantially reduced by the TPL-2 S443A mutation (Fig. 5D). and LPS stimulation of ERK-1/2 in Map3k8 / BMDMs. These Furthermore, S443A mutation prevented TPL-2 rescuing LPS in- results suggested that 14-3-3 binding constitutively to phospho- − − duction of ERK-1/2 following retroviral transduction of Map3k8 / S400 or to phospho-S443 was not sufficient to fully activate TPL-2. BMDMs (Fig. 5F). Immunoblotting with phospho-S443 antibody revealed that IL-1β – To determine whether 14-3-3 binding to phospho-S443 was stimulation induced S443 phosphorylation on TPL-2S400 R18 – sufficient to promote TPL-2 signaling, the S443 14-3-3 binding (Fig. 6C) and S400 phosphorylation on TPL-2S443 R18 (Fig. 6D). site in TPL-2 was replaced with the R18 motif. Introduction of Because 14-3-3 dimers are able to bind separately to two phos- the R18 peptide into the S443 binding site did not alter the basal phopeptide motifs, these findings implied that a 14-3-3 dimer catalytic activity of TPL-2 (Fig. S5A), but did abrogate its ability bound simultaneously to phospho-S400 and -S443 to promote – – to interact with p105 (Fig. S5B). TPL-2S443 R18 was able to bind TPL-2 signaling. Consistent with this possibility, TPL-2S400 R18/S443A – to 14-3-3 constitutively, but mediated activation of MEK-1/2 and TPL-2S443 R18/S400A, although constitutively bound to 14-3-3, in IL-1R–293 cells only after IL-1β stimulation (Fig. 6A). LPS failed to mediate LPS activation of ERK-1/2 when expressed in − − – – stimulation was also required to induce ERK-1/2 phosphorylation Map3k8 / BMDMs, in contrast to TPL-2S400 R18 and TPL-2S443 R18, − − – in Map3k8 / BMDMs expressing TPL-2S443 R18 (Fig. 6B). Thus, respectively (Fig. 6E). Together, these results indicated that IMMUNOLOGY

Fig. 6. Optimal TPL-2 signaling requires 14-3-3 interaction with both phospho-S400 and -S443. (A) IL-1R–293 cells expressing the indicated Myc–TPL-2 − − constructs or empty vector (EV) were stimulated with IL-1β or left unstimulated. GST–14-3-3γ pulldowns and lysates were immunoblotted. (B and E) Map3k8 / BMDMs, transduced with retroviruses encoding the indicated TPL-2 proteins, were stimulated with LPS for 15 min or left unstimulated. Lysates were immunoblotted. (C and D) WT and mutant forms of Myc–TPL-2 were immunoprecipitated from lysates of IL-1R–293cellswithorwithoutIL-1β stimu- lation, and immunoblotted. (F)IL-1R–293 cells expressing the indicated Myc–TPL-2 constructs were stimulated with IL-1β or left unstimulated. Lysates were immunoblotted.

Ben-Addi et al. PNAS | Published online May 27, 2014 | E2399 Downloaded by guest on September 30, 2021 TPL-2 must be phosphorylated on both S400 and S443 to effi- these results indicated that 14-3-3 binding to the phosphorylated C ciently recruit 14-3-3 dimers and trigger activation of ERK-1/2 terminus augmented TPL-2 catalytic activity toward MEK-1. MAP kinases. TPL-2S400A mutation completely blocked the phosphorylation of To investigate whether 14-3-3 binding to both phospho-S400 endogenous MEK-1/2 in LPS-stimulated macrophages (Fig. 1B). and -S443 was sufficient to fully activate TPL-2 signaling, we In contrast, immunoprecipitated TPL-2S400A phosphorylated generated a TPL-2 chimera in which the R18 peptide was inserted GST–MEK-1 to a similar degree to WT TPL-2, although only the into both S400 and S443 sites. Expression of this chimera in IL-1R– latter was activated by 14-3-3 binding (Figs. 2A and 7B). For in 293 cells slightly increased basal phospho–MEK-1/2 levels, and this vitro kinase assays, the concentration of GST–MEK-1 was near to β F increase was further augmented by IL-1 stimulation (Fig. 6 ). the Km of TPL-2 for MEK-1 (38). Endogenous MEK-1 is likely Introduction of two positively charged residues in the S400–R18 present in much lower concentration in cells, suggesting that sequence to disrupt 14-3-3 binding to this site blocked both basal phospho-S400 recruitment of 14-3-3 might only be essential for and IL-1β–induced MEK-1/2 phosphorylation. These results in- TPL-2 signaling activity at low concentrations of MEK-1/2 sub- dicated that 14-3-3 must bind simultaneously to both S400 and strate. To investigate this possibility, the ability of Myc–TPL-2S400A S443 sites to promote TPL-2 signaling. However, maximal ac- to phosphorylate overexpressed HA–MEK-1 was tested in IL-1R– tivation of MEK-1/2 by TPL-2S400R18/S443R18 still required IL-1β 293 cells. In contrast to endogenous MEK-1/2 (Fig. 3C), Myc– stimulation, suggesting the existence of an additional 14-3-3– TPL-2S400A was still able to phosphorylate overexpressed HA– independent activating step. MEK-1 after IL-1β stimulation, although not to the same degree as WT Myc–TPL-2 (Fig. 7C). The binding of 14-3-3 to TPL-2, Binding of 14-3-3 Does Not Promote TPL-2 Dimerization. A key step therefore, was not essential for phosphorylation of MEK-1 when in the activation of the MEK kinase B-Raf involves its hetero- the latter protein was expressed at supraphysiological levels. dimerization with Raf-1 (33, 34). This interaction is dependent Together, these data indicate that binding of 14-3-3 dimers to on 14-3-3 binding, which stabilizes the active conformation of the TPL-2 C terminus increases the efficiency of TPL-2 substrate B-Raf (35). Earlier studies have suggested that TPL-2 can phosphorylation, which is essential for TPL-2 phosphorylation of also dimerize (36). Because 14-3-3 bound to two phosphorylated MEK-1/2 at the limiting concentrations present in cells. residues within the TPL-2 C terminus, it was not clear whether TPL-2 dimerization could be mediated via 14-3-3 dimers. To Discussion – – investigate this question, Flag TPL-2 and V5 TPL-2 were co- Previous studies established that TPL-2 activation of MEK-1/2 in – expressed in IL-1R 293 cells. Immunoblotting of anti-Flag im- TLR-stimulated macrophages requires its release from its asso- – – munoprecipitates demonstrated that Flag TPL-2 and V5 TPL-2 ciated inhibitor NF-κB1 p105, which is triggered by IKK2- A heterodimerize (Fig. S7 ). This association was not affected induced proteolysis of p105 by the proteasome (17, 18, 22). Here, β either by IL-1 stimulation (to promote TPL-2 S400/S443 phos- we demonstrate that IKK2 controls a second critical step in TPL-2 phorylation and association with 14-3-3) or S400A mutation (to activation of the ERK-1/2 MAP kinase pathway by directly block TPL-2/14-3-3 interaction), indicating that TPL-2 dimeriza- – phosphorylating the TPL-2 C terminus (21). IKK2 phosphory- tion was independent of 14-3-3 interaction. Pulldowns with GST lation of S400, together with autophosphorylation on S443, MEK-1 also demonstrated that S400A mutation did not affect B triggers recruitment of 14-3-3 dimers to the TPL-2 C terminus. binding of TPL-2 to MEK-1 (Fig. S7 ), which is required for This interaction facilitates TPL-2 phosphorylation of MEK-1/2, efficient MEK-1/2 phosphorylation by TPL-2 (12). The 14-3-3 possibly by relieving the inhibitory interaction between the TPL-2 interaction, therefore, did not facilitate TPL-2 activation of MEK- C terminus and kinase domain (37). 1/2 by promoting TPL-2 binding to MEK-1 or itself. By replacing the sequence surrounding S400 with R18 motif – to generate TPL-2S400 R18, it was possible to demonstrate that Binding of 14-3-3 Enhances TPL-2 Catalytic Activity. Truncation of the C terminus activates the oncogenic potential of TPL-2, which TPL-2 activation can occur in the absence of S400 phosphorylation, – provided that 14-3-3 binding to the C terminus was maintained. correlates with an increase in TPL-2 specific kinase activity (37). S400–R18KK Furthermore, the free C terminus can bind in trans to the TPL-2 Importantly, TPL-2 , which could not interact with 14-3- kinase domain, reducing its catalytic activity toward several 3, was not able to activate ERK-1/2 after agonist stimulation, model substrates. These data suggest that TPL-2 catalytic activity ruling out signaling due to structural alterations in the C terminus might be negatively regulated by an intramolecular interaction caused by insertion of the R18 sequence. Similarly, inserting the involving its C terminus. The 14-3-3 dimers are rigid structures R18 peptide sequence in to the sequence surrounding S443 and can induce conformational changes in protein ligands (29). allowed TPL-2 to interact with 14-3-3 and to activate ERK-1/2 in This characteristic raised the possibility that 14-3-3 binding might the absence of S443 phosphorylation. These data suggest that S400 alter the interaction between the kinase domain and C terminus and S443 do not have alternative functions in TPL-2 signaling other of TPL-2, thereby increasing TPL-2 catalytic activity. than the recruitment of 14-3-3 dimers. The level of ERK-1/2 acti- S400–R18 S443–R18 To investigate this possibility, we tested effect of recombinant vation in cells expressing TPL-2 or TPL-2 was always 14-3-3 protein on the ability of TPL-2 to phosphorylate MEK-1 lower than WT TPL-2, suggesting that the R18 peptide did not fully in vitro. The addition of 14-3-3ζ to immunoprecipitates of Flag– recapitulate the effects of the phosphorylated S400 and S443 14-3-3 TPL-2 from IL-1β–stimulated IL-1R–293 cells increased phos- binding sites. This observation may be due to differences in the way phorylation of GST–MEK-1 by approximately fivefold (Fig. 7A). that 14-3-3 bound to the R18 motif compared with the phospho- GST–MEK phosphorylation was further enhanced by coex- S400 or -S443 peptides or because the spacing between 14-3-3 and pression of Flag–TPL-2 with IKK2, which increased both S400 the kinase domain is slightly compromised in the R18 fusions and S443 phosphorylation in combination with IL-1β stimula- compared with the WT molecule. Similarly, replacement of the tion. In contrast, the addition of 14-3-3ζ had no detectable effect S621 14-3-3 binding site in Raf-1 with R18 peptide only partially on the MEK-1 kinase activity of Flag–TPL-2S400A, isolated from rescues Raf-1 signaling activity (39). IL-1β–stimulated cells coexpressing IKK2. Consistent with these S443 resides within the kinase regulatory domain of the TPL-2 data, the addition of recombinant 14-3-3ζ to TPL-2 immuno- C terminus (40), suggesting that binding of 14-3-3 to this region precipitated from LPS-stimulated WT primary macrophages in- might alleviate its regulatory activity. However, agonist stimula- – creased the phosphorylation of GST–MEK-1 (approximately tion was required for TPL-2S443 R18 to induce MEK-1/2 and threefold), whereas 14-3-3ζ had no effect on the MEK kinase ERK-1/2 activation, indicating that 14-3-3 binding to the kinase activity of immunoprecipitated TPL-2S400A (Fig. 7B). Together, regulatory domain was insufficient to activate TPL-2 signaling.

E2400 | www.pnas.org/cgi/doi/10.1073/pnas.1320440111 Ben-Addi et al. Downloaded by guest on September 30, 2021 PNAS PLUS

Fig. 7. TPL-2 MEK kinase activity is enhanced by 14-3-3. (A) IL-1R–293 cells, transiently coexpressing Flag–TPL-2 or Flag–TPL-2S400A and HA–IKK2, were stimulated with IL-1β. Immunoprecipitated Flag–TPL-2 was assayed for its ability to phosphorylate GST–MEK-1K207A (KA) with or without recombinant 14-3-3ζ protein. (B) Endogenous TPL-2 was immunoprecipitated from lysates of BMDMs with or without LPS (15 min) and assayed for its ability to phosphorylate GST– MEK-1K207A (KA) with or without recombinant 14-3-3ζ protein. (C) IL-1R–293 cells were transfected with the indicated amounts of HA–MEK-1 plasmid to- gether with a fixed quantity of Myc–TPL-2 plasmid. Lysates, prepared with or without IL-1β stimulation, were immunoblotted.

– The agonist-inducible phosphorylation of S400 in the TPL-2S443 R18 Consequently, C-terminal truncation of TPL-2 results in un- – chimera, together with the failure of TPL-2S443 R18/S400A to activate controlled phosphorylation of downstream substrates, promoting MEK-1/2 following IL-1β stimulation, indicates that 14-3-3 dimers cell transformation. must bind to both pS400 and pS443 to promote TPL-2 signaling. In conclusion, our study supports a model in which the cata- In line with this suggestion, S443 was inducibly phosphorylated lytic activity of TPL-2 is inhibited in nonstimulated cells by the – – on TPL-2S400 R18,andTPL-2S400 R18/S443A was unable to activate interaction of the TPL-2 C terminus with the kinase domain. MEK-1/2 after IL-1β stimulation. It therefore appears that 14-3-3 TLR stimulation induces IKK phosphorylation of TPL-2 S400 dimers must bind to the TPL-2 C terminus in a specific orientation and TPL-2 S443 autophosphorylation, which triggers the re- to activate TPL-2 signaling to ERK-1/2. Interestingly, TPL-2 S443 cruitment of 14-3-3 to the TPL-2 C terminus. The 14-3-3 binding autophosphorylation in IL-1R–293 cells, which was independent of increases TPL-2–specific activity for MEK-1, possibly by altering IKK2 and S400 phosphorylation, required IL-1β stimulation. Thus, the interaction of the C terminus with the TPL-2 kinase domain an additional, as yet uncharacterized, agonist-induced regulatory (37), which is essential for TPL-2 activation of ERK-1/2 in cells. step triggers TPL-2 S443 autophosphorylation. Furthermore, IL-1β TPL-2 activation of MEK-1/2 also requires its release from NF- stimulation was required for maximal activation of MEK-1/2 by κB1 p105 inhibition, which results from IKK-induced p105 pro- TPL-2S400R18/S443R18, suggesting that agonist stimulation controls teolysis by the proteasome (17, 18). The IKK complex therefore TPL-2 signaling independently of S443 phosphorylation and 14- directly controls two key steps in the activation of TPL-2 by 3-3 binding to the C terminus of TPL-2. TLRs, highlighting the close linkage between ERK-1/2 MAP TPL-2 can induce sTNF production by LPS-stimulated mac- kinase and NF-κB activation in inflammation. rophages independently of its IKK-induced release from p105 and ERK-1/2 activation (22). Map3k8S400A mutation blocked Materials and Methods LPS induction of TNF by BMDMs and prevented the inter- Mouse Strains. Mouse strains were bred in the specific pathogen-free animal action of p105-associated TPL-2 with GST–14-3-3. Further- facility of the National Institute for Medical Research (London). Seven- to 12- S400–R18 week-old mice were used for all experiments, which were performed in more, expression of TPL-2 rescued sTNF production − − Map3k8−/− accordance with the United Kingdom Home Office regulations. Nfkb1 / (42) by LPS-stimulated BMDMs. These data indicate −/− that phosphorylation of the unknown target protein(s) by which and Map3k8 (5) mouse strains have been described. The generation of Map3k8S400A/S400A mice is detailed in SI Materials and Methods. All mouse IMMUNOLOGY p105-associated TPL-2 controls sTNF production is dependent strains were fully back-crossed onto a C57BL/6 background. on 14-3-3 interaction with the TPL-2 C terminus. Map3k8S400A mutation also blocked ERK-1/2 activation in macrophages by all Reagents. Myc-TPL-2, Flag-TPL-2D270A, and Flag-TPL-2 subcloned in pCDNA3 κ tested TLR agonists and TNF. Activation of NF- B in Jurkat T vector have been described (12, 43). Expression constructs encoding Flag and cells by overexpressed TPL-2 is also blocked by S400A mutation V5 epitope-tagged versions of WT and mutant TPL-2 and StepII-p105 were (26). The 14-3-3 binding appears to be required for TPL-2 to generated by PCR, using a Phusion polymerase kit (Finnzyme). All plasmids activate all downstream signaling pathways. were verified by DNA sequencing. Activation of the oncogenic potential of TPL-2 requires de- Antibodies to TPL-2 (M20 and H-7) and ERK-1/2 were obtained from letion of its C terminus (37, 41). Our work suggests that one of Santa Cruz. Antibody to activated phospho(T185/Y187)-ERK-1/2 (P-ERK) was the major effects of C-terminal deletion is to disrupt the normal obtained from Biosource. Antibodies against p38, phospho(T180/Y182)-p38 – regulatory mechanisms that control TPL-2 signaling activity. The (P-p38), phospho MEK-1/2, Flag, and 14-3-3 were purchased from Cell Sig- recruitment of 14-3-3 dimers to the phosphorylated C terminus naling Technology. Phospho-S400 TPL-2 antibody has been described (20). To generate the phospho-S443–TPL-2 antibody, a peptide was synthesized to of WT TPL-2, which increases its specific activity for MEK-1, is correspond to residues 437–449 of murine TPL-2 in which S443 was phos- essential for TPL-2 activation of ERK-1/2 in cells. Removal of phorylated. This phosphopeptide was coupled to keyhole limpet hemocya- the C terminus renders TPL-2 kinase activity independent of a nin and injected into rabbits. An unphosphorylated form of the immunizing requirement for 14-3-3 binding and also disrupts the negative peptide (10 μg/mL) was incubated with phospho-S443 antibody during im- regulatory effects of p105 on TPL-2 MEK kinase activity (12). munoblotting to minimize recognition of unphosphorylated TPL-2.

Ben-Addi et al. PNAS | Published online May 27, 2014 | E2401 Downloaded by guest on September 30, 2021 GST–14-3-3γ was expressed at 30 °C in Escherichia coli BL21 (DE3) and used for pulldowns of StepII–p105. Isolated proteins were eluted from beads purified by affinity chromatography on glutathione–Sepharose 4B (Amer- with 2x SDS/PAGE sample buffer and analyzed by immunoblotting. sham Biosciences). Protein purity was estimated to be >90% by Coomassie brilliant blue (Novex) staining after SDS/PAGE. Recombinant His–14-3-3ζ was TPL-2 MEK Kinase Assays. BMDMs or IL-1R–293 cells were cultured for 18 h in produced as described (44). 1% or 0% serum, respectively. Cells were stimulated by 100 ng/mL LPS for LPS (Salmonella enterica serovar Minnesota R595) was purchased from 15 min (BMDMs) or 20 ng/mL hIL-1β for 20 min (IL-1R–293 cells) or left Alexis Biochemicals. CpG ODN 1668, Pam3Cys4, Imiquimod, and Flagellin unstimulated and lysed by using kinase assay lysis buffer (buffer A con- were obtained from Invivogen. Recombinant mouse TNF and human IL-1β taining 0.5% Nonidet P-40 and 1 mM DTT). Endogenous TPL-2 was immu- were from Peprotec. Phorbol 12-myristate 13-acetate was bought from noprecipitated from BMDM lysates by using a 1:1 mixture of 70-mer (17) and Sigma. Inhibitors BI605906 (45) and C34 (32) were provided by the MRC M20 (Santa Cruz) TPL-2 antisera coupled to protein A Sepharose. Flag mAb Protein Phosphorylation Unit (University of Dundee, Scotland). affinity gel (Sigma) was used to immunoprecipitate transfected Flag–TPL-2. Immunoprecipitates were washed four times in kinase assay lysis buffer and In Vitro Generation of Macrophages. BMDMs were prepared as described twice in kinase buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM β-glycer- (17, 46). More than 95% of the resulting BMDM cell populations were ophosphate, 100 nM okadaic acid, 1 mM DTT, 0.1 mM sodium vanadate, 10 + + F4/80 CD11b . Before stimulation, cells were cultured overnight in medium mM MgCl2, 1 mM EGTA, and 0.03% Brij 35). Beads were then resuspended in containing reduced FBS (1%) and no L-cell conditioned medium. 50 μL of kinase buffer plus 1 mM ATP and 0.5 μM kinase-inactive GST–MEK- 1D208A protein (MRC Protein Phosphorylation Unit, Dundee University) or GST– Retrovirus Infection of Macrophages. Amphoteric recombinant retroviruses MEK-1K207A (49). After 30 min at 30 °C, reactions were terminated by the addi- − − were produced as described (17). For retroviral infection, Map3k8 / or tion of EDTA. Phosphorylation of GST–MEK-1D208A and GST–MEK-1K207A were − − Nfkb1 / BM cells were plated in complete BMDM medium [RPMI medium assessed by immunoblotting using phospho–MEK-1 mAb (Cell Signaling). 1640 (Sigma) supplemented with 10% (vol/vol) heat-inactivated FBS, 20% (vol/vol) L-cell conditioned medium, 2 mM glutamine, nonessential TNF Assay. Concentrations of TNF in culture supernatants and sera were amino acids, antibiotics, 10 mM Hepes, and 50 μM β-mercaptoethanol] at 1 × determined by using commercial ELISA kits (eBioscience). 106 cells per well of six-well plate (2 mL of culture volume; Sarstedt). Fol- μ lowing 4 d of culture, 200 L of virus containing supernatant was added per In Vivo Analysis of ERK-1/2 Activation. In vivo analysis of ERK-1/2 phosphor- × well, and plates were centrifuged at 2,000 g for 1 h. Cells were cultured for ylation in macrophages was performed as described (22). 3 h, 4 mL of complete BMDM medium was then added, and cells were recultured for a further 4 d. Cells harvested at this time were >95% F4/80+. Biolayer Interferometry. Binding of 14-3-3 to peptides was measured on an For experiments, cells were replated at 1 × 106 cells per well (2 mL of culture Octet RED biolayer interferometer (Pall ForteBio Corp.). Biotinylated phos- volume) of a six-well plate (Nunc) in RPMI medium plus 1% FBS and lacking pho-S621 Raf peptide (at 1.5 μg/mL) was immobilized on streptavidin bio- L-cell conditioned medium. sensors (Pall ForteBio Corp.). These sensors were then exposed to different concentrations of 14-3-3ζ (0.05–5 μM), and the response was recorded at the The 293 Cell Culture and Transfection. HEK-293 cells stably expressing the IL-1R end of a 10-min association step. All measurements were made at 25 °C in 10 (C6 cells) were provided by Xiaoxia Li (Cleveland Clinic Foundation, Cleveland) mM sodium phosphate, 150 mM NaCl, 0.002% Tween 20, 0.005% sodium (47). C6 cells were transiently transfected by using Lipofectamine 2000 (Life azide, and 0.1 mg/mL BSA. The dependence of the response on the 14-3-3 ζ Technologies). After overnight culture in complete medium [Dulbecco’s concentration was analyzed by using the standard nonlinear least-squares modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% methods to calculate the K for phospho-S621 Raf peptide binding (1:1 (vol/vol) FCS and 10 mM Hepes], cells were serum-starved for 24 h before d binding model). stimulation with recombinant IL-1β (Peprotech; 20 ng/mL) for 20 min. Equilibrium dissociation constants for the nonbiotinylated TPL-2 peptides were determined by using competition assays in which phospho-S621 Raf Protein Analyses. Cells were washed once in cold PBS before lysis. For im- peptide-loaded sensors were exposed to a solution of 14-3-3ζ (at 1 μM) munoblotting, cells were lysed in buffer A [50 mM Tris, pH 7.5, 150 mM NaCl, containing different concentrations of the competing peptide (0.05–80 μM). 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na VO , 100 nM okadaic acid 3 4 Standard nonlinear least-squares methods were used to calculate the K for (Calbiochem), 2 mM Na P O , and 5 mM Na β-glycerophosphate plus pro- d 4 2 7 the competing peptide with the K for phospho-S621 Raf peptide binding tease inhibitors (Roche Molecular Biochemicals)] containing 1% Nonidet- d (0.49 ± 0.08 μM) fixed in the analysis (Fig. S6C). P40, 0.5% deoxycholate, and 0.1% SDS. After centrifugation, lysates were mixed with an equal volume of 2x SDS/PAGE sample buffer. For immuno- precipitations, lysis was carried out by using buffer B [50 mM Hepes, pH 7.5, Statistical Analysis. In vitro data were compared by using the Student t test 1 mM EDTA, 20 mM NaF, and 1 mM Na P O plus a mixture of protease (two-tailed and unpaired test). For in vivo experiments, all statistical com- 4 2 7 – inhibitors (Roche Molecular Biochemicals)] containing 0.5% Nonidet-P40. parisons were carried out by using the nonparametric two-tailed Mann Covalent coupling of antibodies to protein A-Sepharose (Amersham Bio- Whitney test. Statistically significant differences are indicated on the figures. sciences) and immunoprecipitation were performed as described (48). To detect release of TPL-2 from p105, BMDM lysates were precleared of p105 by ACKNOWLEDGMENTS. We thank the National Institute for Medical Research anti-p105 immunodepletion before immunoblotting (17). (NIMR) Photographics department, NIMR Biological Services, NIMR flow cytometry service, Caroline Morris (NIMR), John Offer (NIMR), Emilie Jacque For pulldown assays, cells were lysed in buffer B plus 0.5% Nonidet P-40. – γ – (NIMR), and members of the Ley laboratory for help during the course of this Recombinant GST, GST 14-3-3 ,orGSTMEK-1 (Millipore) proteins were work. We also thank Victor Tybulewicz (NIMR) and Peter Parker (Cancer μ – mixed with lysates plus 10 L of packed glutathione Sepharose 4B beads Research UK, London) for helpful comments on the manuscript. This study (Amersham Biosciences) and incubated for 4h at 4 °C. Beads were then was supported by UK Medical Research Council Grant U117584209, Arthritis washed in lysis buffer B. Steptactin–Sepharose (Amersham Biosciences) was Research UK Grant 18864 and Leukaemia Research Fund Grant 06050.

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