The Scaffold MyD88 Acts to Couple Protein Kinase Cϵ to Toll-like Receptors
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.m710330200
ISSN1083-351X
AutoresAmir Faisal, Adrian T. Saurin, Bernard Gregory, Brian M. J. Foxwell, Peter J. Parker,
Tópico(s)interferon and immune responses
ResumoMice lacking protein kinase Cϵ (PKCϵ) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCϵ coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCϵ involvement in TLR signaling and found that PKCϵ is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCϵ binding to 14-3-3β. LPS-induced PKCϵ phosphorylation, 14-3-3β binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCϵ phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCϵ association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCϵ. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCϵ was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCϵ at these two sites is required for TLR4- and TLR2-induced NFκB reporter activation and IκB degradation in reconstituted PKCϵ–/– cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCϵ recruitment, phosphorylation, and downstream signaling. Mice lacking protein kinase Cϵ (PKCϵ) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCϵ coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCϵ involvement in TLR signaling and found that PKCϵ is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCϵ binding to 14-3-3β. LPS-induced PKCϵ phosphorylation, 14-3-3β binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCϵ phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCϵ association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCϵ. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCϵ was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCϵ at these two sites is required for TLR4- and TLR2-induced NFκB reporter activation and IκB degradation in reconstituted PKCϵ–/– cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCϵ recruitment, phosphorylation, and downstream signaling. Toll-like receptors (TLRs) 2The abbreviations used are: TLR, Toll-like receptor; PKC, protein kinase C; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; WT, wild type; MEF, mouse embryo fibroblasts; GFP, green fluorescent protein; IRF, interferon regulatory factor; TIR, Toll-IL1-R; GST, glutathione S-transferase; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; siRNA, short interfering RNA; YFP, yellow fluorescent protein; RANTES, regulated on activation normal T cell expressed and secreted; TRIF, Toll-IL-1R domain-containing adaptor inducing interferon-β; TRAM, TRIF-related adaptor molecule; PIP2, phosphatidylinositol 4,5-bisphosphate; LTA, lipoteichoic acid. recognize microbial pathogen-associated molecular patterns and initiate common signaling pathways leading to specific inflammatory responses through activation of transcription factors such as nuclear factor κB (NFκB) and interferon regulatory factors (IRFs) (1Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8899) Google Scholar). TLRs signal through Toll-IL1-R (TIR) domain-containing adaptor proteins that are recruited to receptor TIR domains upon ligand binding (2Miggin S.M. O'Neill L.A. J. Leukocyte Biol. 2006; 80: 220-226Crossref PubMed Scopus (224) Google Scholar). Of five TIR domain-containing adaptors identified in humans, MyD88 has been shown to be involved in signal transduction for all TLRs except TLR3 (3Jiang Z. Zamanian-Daryoush M. Nie H. Silva A.M. Williams B.R. Li X. J. Biol. Chem. 2003; 278: 16713-16719Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 4O'Neill L.A. Bowie A.G. Nat. Rev. 2007; 7: 353-364Google Scholar). MyD88 deficiency in macrophages and dendritic cells leads to loss of MAPK activation, NFκB activation, and proinflammatory cytokine production in response to various TLR ligands (5Hayashi F. Smith K.D. Ozinsky A. Hawn T.R. Yi E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2837) Google Scholar, 6Hemmi H. Kaisho T. Takeuchi O. Sato S. Sanjo H. Hoshino K. Horiuchi T. Tomizawa H. Takeda K. Akira S. Nat. Immunol. 2002; 3: 196-200Crossref PubMed Scopus (2081) Google Scholar, 7Schnare M. Holt A.C. Takeda K. Akira S. Medzhitov R. Curr. Biol. 2000; 10: 1139-1142Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). However, some responses downstream of TLR4 are either only delayed (NFκB and MAPK activation) or not affected (INFβ production) in MyD88-deficient cells (8Kawai T. Takeuchi O. Fujita T. Inoue J. Muhlradt P.F. Sato S. Hoshino K. Akira S. J. Immunol. 2001; 167: 5887-5894Crossref PubMed Scopus (903) Google Scholar). These constitute MyD88-independent pathways and have led to the identification of other adaptor proteins. MyD88 adaptor-like (Mal) and Toll-IL-1R domain-containing adaptor inducing interferon-β (TRIF)-related adaptor molecule (TRAM) work as bridging adaptors for MyD88 and TRIF to activate NFκB and IRF3, respectively. Mal/MyD88 signal from TLR2/TLR4 to regulate NFκB activation (MyD88-dependent), whereas TRAM/TRIF signal to IRF3 in response to TLR4 activation (MyD88-independent) (9Sheedy F.J. O'Neill L.A. J. Leukocyte Biol. 2007; 82: 196-203Crossref PubMed Scopus (55) Google Scholar). The specificity in the activation of transcription factors by different TLRs using common signaling pathways is therefore achieved by differential use of adaptor proteins. Protein kinase C (PKC) is a family of closely related serine/threonine kinases that regulate diverse cellular processes such as proliferation, survival, immunity, and apoptosis (10Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1361) Google Scholar, 11Griner E.M. Kazanietz M.G. Nat. Rev. Cancer. 2007; 7: 281-294Crossref PubMed Scopus (787) Google Scholar). Based on the cofactor requirements, the PKC family is classified into three subfamilies as follows: conventional PKCs (α, βI, βII, and γ) regulated by diacylglycerol, phosphatidylserine, and calcium; novel PKCs (δ, ϵ, θ, and η) regulated by diacylglycerol and phosphatidylserine; and atypical PKCs (ζ and λ/ι) that do not require diacylglycerol for activation (12Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar). Studies in mice lacking different PKC isoforms have established an important role for PKCs in intracellular immune signaling (reviewed in Ref. 13Tan S.L. Parker P.J. Biochem. J. 2003; 376: 545-552Crossref PubMed Scopus (212) Google Scholar). PKCβ knock-out mice have an immunodeficiency because of defective B cell activation (14Leitges M. Schmedt C. Guinamard R. Davoust J. Schaal S. Stabel S. Tarakhovsky A. Science. 1996; 273: 788-791Crossref PubMed Scopus (413) Google Scholar), whereas PKCθ is required for T cell receptor-mediated T cell activation (15Sun Z. Arendt C.W. Ellmeier W. Schaeffer E.M. Sunshine M.J. Gandhi L. Annes J. Petrzilka D. Kupfer A. Schwartzberg P.L. Littman D.R. Nature. 2000; 404: 402-407Crossref PubMed Scopus (792) Google Scholar). Mice deficient in PKCδ and PKCζ have defective B cell anergy and NFκB signaling, respectively (16Leitges M. Sanz L. Martin P. Duran A. Braun U. Garcia J.F. Camacho F. Diaz-Meco M.T. Rennert P.D. Moscat J. Mol. Cell. 2001; 8: 771-780Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 17Mecklenbrauker I. Saijo K. Zheng N.Y. Leitges M. Tarakhovsky A. Nature. 2002; 416: 860-865Crossref PubMed Scopus (235) Google Scholar). PKCϵ–/– mice have impaired innate immunity and fail to clear Gram-positive and Gramnegative bacterial infection (18Castrillo A. Pennington D.J. Otto F. Parker P.J. Owen M.J. Bosca L. J. Exp. Med. 2001; 194: 1231-1242Crossref PubMed Scopus (205) Google Scholar). LPS and INFγ-induced nitric oxide, tumor necrosis factor-α, IL1β, and prostaglandin E2 production in PKCϵ–/– macrophages is reduced, and this is attributed to the impaired NFκB activation upstream of IκB kinase β. Other PKC isoforms have also been implicated in TLR signaling. PKCα is involved in LPS- and poly(I-C)-induced NF-IL6 (19Chano F. Descoteaux A. Eur. J. Immunol. 2002; 32: 2897-2904Crossref PubMed Scopus (8) Google Scholar) and IRF3 (20Johnson J. Albarani V. Nguyen M. Goldman M. Willems F. Aksoy E. J. Biol. Chem. 2007; 282: 15022-15032Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) activation, respectively. LPS-induced MAPK activation, tumor necrosis factor-α production, and NFκB activation were shown to be PKCζ-dependent in different cell types (21Cuschieri J. Umanskiy K. Solomkin J. J. Surg. Res. 2004; 121: 76-83Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Dallot E. Mehats C. Oger S. Leroy M.J. Breuiller-Fouche M. Biochimie (Paris). 2005; 87: 513-521Crossref PubMed Scopus (38) Google Scholar, 23Monick M.M. Carter A.B. Flaherty D.M. Peterson M.W. Hunninghake G.W. J. Immunol. 2000; 165: 4632-4639Crossref PubMed Scopus (112) Google Scholar). Similarly, involvement of PKCϵ in MKP-1 and IL12 induction in response to LPS stimulation in macrophages and dendritic cells, respectively, has been demonstrated (24Aksoy E. Amraoui Z. Goriely S. Goldman M. Willems F. Eur. J. Immunol. 2002; 32: 3040-3049Crossref PubMed Scopus (45) Google Scholar, 25Valledor A.F. Xaus J. Comalada M. Soler C. Celada A. J. Immunol. 2000; 164: 29-37Crossref PubMed Scopus (100) Google Scholar). Despite many studies implicating PKC isoforms in TLR signaling, there is little evidence on the mechanism of their involvement. Recently, TRAM has been identified as a substrate of PKCϵ, and its phosphorylation has been shown to regulate RANTES production through IRF3 activation (26McGettrick A.F. Brint E.K. Palsson-McDermott E.M. Rowe D.C. Golenbock D.T. Gay N.J. Fitzgerald K.A. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9196-9201Crossref PubMed Scopus (109) Google Scholar). However, the mechanism of PKCϵ activation in response to LPS remains obscure, and although TRAM phosphorylation by PKCϵ was required for it to signal, the exact function of this phosphorylation remains elusive. Similarly, Kubo-Murai et al. (27Kubo-Murai M. Hazeki K. Sukenobu N. Yoshikawa K. Nigorikawa K. Inoue K. Yamamoto T. Matsumoto M. Seya T. Inoue N. Hazeki O. Mol. Immunol. 2007; 44: 2257-2264Crossref PubMed Scopus (41) Google Scholar) have shown recently that PKCδ binds to Mal, and this binding promotes TLR2 and TLR4 signaling to p38 MAPK and IκB. We have recently identified novel phosphorylation sites (Ser-346 and Ser-368) in the V3 region of PKCϵ that regulate its association with 14-3-3β. 3A. T. Saurin, J. Durgan, A. J. Cameron, A. Faisal, M. S. Marber, and P. J. Parker (2008) Nat. Cell. Biol., in press. Phosphorylation at these sites occurs sequentially through p38 (Ser-350) followed by GSK-3β (Ser-346) and auto-phosphorylation or classic PKC trans-phosphorylation (Ser-368) (43Durgan J. Cameron A.J. Saurin A.T. Hanrahan S. Totty N. Messing R.O. Parker P.J. Biochem. J. 2008; 411: 319-331Crossref PubMed Scopus (30) Google Scholar). The subsequent binding of 14-3-3β is required for efficient separation of cells at the end of cytokinesis. Here we investigated the role of PKCϵ in TLR signaling and discovered that LPS induced both recruitment of PKCϵ to TLR4 and its phosphorylation at Ser-346 and Ser-368, resulting in its association with 14-3-3β. PKCϵ recruitment to TLR4, phosphorylation, and binding to 14-3-3β were all dependent on MyD88 expression. We therefore propose that MyD88 represents the missing link that couples PKCϵ to TLR4 in response to LPS. Reagents—Lipopolysaccharide (L7261) and lipoteichoic acid (L2515) were purchased from Sigma, and other TLR ligands were from Invivogen. All the inhibitors except BIRB 796 (a gift from Dr. Ana Cuenda, Dundee, Scotland, UK) were from Calbiochem. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies, ECL reagent, glutathione-Sepharose, and protein G-Sepharose were from Amersham Biosciences. The dual luciferase reporter system was purchased from Promega. Rabbit polyclonal antibodies against PKCϵ (C-15) and MyD88 (HFL-296) were from Santa Cruz Biotechnology. Mouse monoclonal anti-FLAG-M2 antibodies were obtained from Sigma. Mouse monoclonal Anti-GFP antibodies 3E1 (for Western blot) and 4E12/8 (for immunoprecipitation) were from the London Research Institute monoclonal facility. Rabbit polyclonal anti-phospho-p38 antibodies were from Cell Signaling. Generation of phospho-specific antibodies to serine 346 and serine 368 were carried out essentially as described previously (28Durgan J. Michael N. Totty N. Parker P.J. FEBS Lett. 2007; 581: 3377-3381Crossref PubMed Scopus (30) Google Scholar) using the immunogens DRSKS(P)APTS and KIT-NS(P)GQRR, respectively. All other reagents were from Sigma. Plasmids—GFP-PKCϵ WT, GFP-PKCϵ regulatory domain, Myc PKCϵ WT, and PKCϵ mutants in pEGFP-C1 and in pCDNA4/TO vectors were constructed by PCR and subcloning and were sequence-verified. The PKCϵ regulatory domain construct was cloned into the pEGFP-C1 vector. The human MyD88 construct was provided by Dr. Shizu Akira. The cDNA was re-cloned into pCDNA 3.1 and pCMV 2B vectors by PCR cloning and then sequence-verified. GST-14-3-3-β was from Professor Alastair Aitken. FLAG- and YFP-tagged TLR (TLR2, -3, and -4) constructs were a kind gift from Professor Golenbock. IRF3-dependent luciferase reporter construct, pGL3–561 (29Grandvaux N. Servant M.J. tenOever B. Sen G.C. Balachandran S. Barber G.N. Lin R. Hiscott J. J. Virol. 2002; 76: 5532-5539Crossref PubMed Scopus (421) Google Scholar), was a kind gift from Dr. Ganes Sen. Cells and Transfections—293 cells stably expressing human TLR4, MD2, and CD14 (referred to as 293/hTLR4) were purchased from Invivogen. These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 10 μg/ml blasticidin, and 50 μg/ml Hygro-Gold at 37 °C in a humidified chamber with 5% CO2. RAW 264.7 cells were maintained in DMEM with 10% FCS at 37 °C in a humidified chamber with 10% CO2. PKCϵ–/– mouse embryo fibroblasts (MEFs) have been described earlier (30Ivaska J. Whelan R.D. Watson R. Parker P.J. EMBO J. 2002; 21: 3608-3619Crossref PubMed Scopus (138) Google Scholar), whereas MEFs from MyD88–/– mice were isolated from 12-day-old embryos and maintained in DMEM with 10% FCS. Peripheral blood monocytes were isolated by elutriation as described elsewhere (31Foxwell B. Browne K. Bondeson J. Clarke C. de Martin R. Brennan F. Feldmann M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8211-8215Crossref PubMed Scopus (240) Google Scholar). Monocytes were differentiated into macrophages for 3 days using 100 ng/ml recombinant human macrophage colony-stimulating factor (PeproTech) in RPMI 1640 medium containing 5% FCS and 100 units/ml penicillin/streptomycin. 293/hTLR4 cells were transfected at ∼80% confluency with Lipofectamine 2000 or LTX (Invitrogen) according to the manufacturer's instructions. For NFκB reporter activation assays, cells were transfected with NFκB-TA-Luc (Clontech) and phRL-Renilla (Promega) at a 10:1 ratio using Lipofectamine LTX. Generation of Stable Cell Lines—∼70% confluent RAW cells in 10-cm plates were transfected with 10 μg/plate of the plasmid DNA (GFP-PKCϵ WT, Ser-346/S368A, S346A, S368A, or vector control) using Lipofectamine 2000. Cells were split into 15-cm plates the next day and selected with 500 μg/ml Zeocin (Invitrogen) or 1 mg/ml G418 (depending on the constructs). Single clones were picked, and the rest were pooled and analyzed for GFP-PKCϵ expression by Western blot. PKCϵ MEFs stably expressing different GFP-PKCϵ constructs were generated by transfection and Zeocin selection of polyclonal populations. 3A. T. Saurin, J. Durgan, A. J. Cameron, A. Faisal, M. S. Marber, and P. J. Parker (2008) Nat. Cell. Biol., in press. siRNA Knockdown—MyD88-N siRNA duplex (Qiagen) targeted the N-terminal region (nucleotides 181–201) of human MyD88 and had the following sequence 5-CCGGCAACUGGAGACACAAdTdT-3 and 5-UUGUGUCUCCAGUUGCCGGdAdT-3. 2 × 105 293/hTLR4 cells in 6-well plates were transfected with 50 nm siRNA using 5 μl of Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. 48 h after transfection with siRNA, cells were re-transfected with GFP-PKCϵ. For experiments investigating PKCϵ-TLR4 interaction after MyD88 knockdown, 60-mm plates were used, and 48 h after MyD88 siRNA transfection cells were co-transfected with GFP-PKCϵ and FLAG-TLR4. Cells were analyzed after a further 24 h. Immunoprecipitation, Pulldown, and Western Blot Analysis—Cell lysis was carried out on ice with lysis buffer containing 1% Nonidet P-40, 50 mm Tris-HCl, pH 7.4, 120 mm NaCl, 5 mm sodium vanadate, 50 mm sodium fluoride, and EDTA-free protease inhibitor tablet from Roche Applied Science. Protein concentration was measured, and equal amounts of protein were immunoprecipitated by incubation at 4 °C with anti-GFP and anti-FLAG antibodies as indicated for 2 h followed by a further 1-h incubation with protein G-Sepharose. Beads were washed twice with TNET (TNE + 1% Triton X-100) and once with TNE (50 mm Tris-HCl, pH 7, 140 mm NaCl, and 5 mm EDTA) and boiled in LDS sample buffer. Proteins were resolved by 4–12% NuPAGE gels, transferred to polyvinylidene difluoride membranes, and immunoblotted with specific antibodies. GST-14-3-3β pulldown assays were performed using bacterially expressed GST-14-3-3β-loaded glutathione beads. Native extracts were tumbled overnight at 4 °C prior to washing twice with TNET (TNE + 1% Triton X-100) and once with TNE (50 mm Tris-HCl, pH 7, 140 mm NaCl, and 5 mm EDTA) and elution in LDS buffer. Isolation of Thioglycollate-elicited Peritoneal Macrophages—Peritoneal macrophages were isolated as described previously (18Castrillo A. Pennington D.J. Otto F. Parker P.J. Owen M.J. Bosca L. J. Exp. Med. 2001; 194: 1231-1242Crossref PubMed Scopus (205) Google Scholar). Briefly, mice (MyD88 knock-out and C57 black/6 WT) were injected with 3% thioglycollate intraperitoneally and sacrificed after 4 days. The peritoneum was flushed with sterilized phosphate-buffered saline (containing 5 mm EDTA), and the peritoneal suspension containing macrophages was carefully removed and centrifuged at 200 × g for 10 min. Cells were resuspended and seeded in RPMI 1640 medium with 10% FCS in 10-cm plates at 37 °C with 10% CO2. After 1 h of incubation, nonadherent cells were removed by extensive washing with phosphate-buffered saline. Cells were used for experiments on the following day. LPS Triggers Phosphorylation of PKCϵ—The defective innate immunity of PKCϵ knock-out mice formally defines PKCϵ as a key regulator of TLR responses. For Gram-negative bacteria the relevant signaling paradigm involves LPS stimulation of TLR4; however, there is little direct evidence that TLR4 activation engages PKCϵ. Recent studies have identified two phosphorylation sites within the V3 region of PKCϵ that enable its binding to 14-3-3β and that are associated with the engagement of PKCϵ in signaling processes.3 To determine whether LPS activation of TLR4 triggers these responses, RAW 264.7 cells (here referred to as RAW cells) stably expressing GFP-PKCϵ were treated with LPS, and phosphorylation of PKCϵ was determined by Western blot using phospho-specific antibodies. Phosphorylation of both the Ser-346 and Ser-368 sites was found to be induced in response to LPS (Fig. 1A); a similar induction was also seen in GFP-PKCϵ-transfected 293 cells stably expressing the human proteins TLR4, MD2, and CD14 (Fig. 1B). There is a degree of constitutive phosphorylation of PKCϵ at the Ser-368 site in both cell types; however, there is clear LPS-dependent induction. LPS also induced Ser-346 phosphorylation of GFP-PKCϵ in HEK 293 cells transiently expressing TLR4/MD2; however, no phosphorylation was observed in vector control transfected cells (data not shown). To test the response of the endogenous PKCϵ in terms of phosphorylation at Ser-346 and Ser-368, use was made of the 14-3-3β interaction of Ser-346/368 doubly phosphorylated PKCϵ (see below). Although this assay does not distinguish whether both sites are induced for the endogenous protein, in light of the observations above it is anticipated that both are increased (serum titers for the site-specific antisera were found to be inadequate for Western blotting the endogenous protein). Pulldown with GST-14-3-3β demonstrated that LPS induced an increased recovery of PKCϵ for both ectopic GFP-PKCϵ and the endogenous PKCϵ (Fig. 1C). A similar pulldown assay was used to demonstrate LPS-induced PKCϵ phosphorylation in primary human macrophages (Fig. 1D). The absolute requirement for dual 346/368 phosphorylation for the 14-3-3β interaction was confirmed through the use of single and double Ser → Ala mutants (Fig. 1E). The LPS-induced responses in RAW cells expressing GFP-PKCϵ were time- and dose-dependent (Fig. 1, F and G). For both the Ser-346 and Ser-368 sites the responses peak at 20–30 min, and both are effectively at basal levels by 2 h. As illustrated (Fig. 1F), the induction of Ser-346 and Ser-368 was >7- and >2-fold, respectively. Over a series of experiments the mean induction for Ser-346 was 7 ± 1.01- and 2.5 ± 0.16-fold for Ser-368. Consistent with the mechanism of Ser-346 phosphorylation in fibroblasts (Ser-350 phosphorylation by p38 primes Ser-346 for phosphorylation by GSK3-β; see below), activation site phosphorylation of p38 MAPK was induced and peaked just ahead of PKCϵ Ser-346 phosphorylation. After 30 min of stimulation, phosphorylation of Ser-346 was induced by as little as 10 ng/ml of LPS and was maximum at 25 ng/ml. LPS Induces PKCϵ Phosphorylation via p38γ/δ and GSK-3—Previous studies in fibroblasts have identified Ser-346 as a target for GSK-3, primed by p38α/β phosphorylation at Ser-350. To determine whether these same pathways account for the LPS-induced response in macrophages, cells were stimulated with LPS and various inhibitors assessed for their effects on Ser-346 phosphorylation. Three different GSK-3 inhibitors were found to block LPS-induced Ser-346 phosphorylation (Fig. 2A and data not shown for SB216763). Inhibition of all four p38 MAPK isoforms (with BIRB 796 (32Kuma Y. Sabio G. Bain J. Shpiro N. Marquez R. Cuenda A. J. Biol. Chem. 2005; 280: 19472-19479Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar)) also blocked Ser-346 phosphorylation indicative of a priming role (Fig. 2B). However, the p38α/β-selective inhibitors failed to block LPS-induced PKCϵ Ser-346 phosphorylation (see further below). Previously, p38α/β have been implicated in UV-induced PKCϵ Ser-346 phosphorylation in fibroblasts.3 Hence the effect of LPS was investigated in MEFs expressing GFP-PKCϵ. As in RAW cells, pan-p38 MAPK inhibition blocked LPS-induced Ser-346 phosphorylation, whereas the p38α/β-selective inhibitors (SB203580 and SB202190) were not inhibitory (Fig. 2C). To confirm the specificity of this behavior, the UV response was re-assessed in MEFs in parallel to the LPS response. As shown in Fig. 2D, BIRB inhibited both the LPS- and UV-induced phosphorylation of PKCϵ at the Ser-346 site. However, whereas SB203580 inhibited the UV-induced response, it did not inhibit the LPS-induced response. These distinct patterns of behavior are indicative of selective activation and/or targeting of specific p38 MAPKs to the same priming phosphorylation of PKCϵ in response to distinct agonists. To assess the expected PKC dependence for Ser-368 phosphorylation, LPS- or LTA (TLR2 ligand; see further below)-stimulated cells were pretreated with BIMI (classic PKC + novel PKC inhibitor). Unexpectedly, no inhibition of Ser-368 phosphorylation was observed even at high BIMI concentration (Fig. 2E), although there was an inhibition by Go6976, a novel PKC inhibitor (data not shown) indicating that a distinct basophilic protein kinase is involved in the LPS response. Thus, although LPS triggers the phosphorylation of the 14-3-3β-binding sites of PKCϵ in both macrophages and fibroblasts, it does so via p38γ/δ + GSK-3 alongside an unknown basophilic kinase. This contrasts with the p38α/β + GSK-3 and PKC-dependent phosphorylation of these sites under other conditions.3 Notwithstanding this notable distinction, it is evident that LPS/TLR4 induce phosphorylation of PKCϵ in line with the established in vivo requirement for this kinase. Multiple TLR Ligands Trigger PKCϵ Phosphorylation—PKCϵ knockout mice are defective in the clearance of both Gram-positive and Gram-negative bacteria. To determine whether model Gram-positive ligands acting via TLR2 promote PKCϵ phosphorylation as determined above for LPS-TLR4, RAW cells were stimulated with either LTA or FSL-1 (TLR2/TLR6). Both ligands were found to increase PKCϵ Ser-346 and Ser-368 phosphorylation (Fig. 3A), although the Ser-368 site was delayed relative to the Ser-346 site and induced only an ∼2-fold increase. In view of the conservation of this response, ligands engaging TLRs 1–9 were tested (Fig. 3B). All but the TLR3 ligand poly(I-C) stimulated PKCϵ Ser-346 phosphorylation. To ensure that poly(I-C) was acting via TLR3, 293/hTLR4 cells were transfected with TLR3 or TLR4, and responses to poly(I-C) and LPS were monitored. LPS-induced phosphorylation of Ser-346 in TLR4 expressing cells was observed, but no such response to poly(I-C) in TLR3-expressing cells was seen (Fig. 3C). However, poly(I-C) did induce an IRF-3-dependent reporter response in TLR3-expressing cells but not in cells expressing TLR4 (Fig. 3D). Thus a functionally linked TLR3 receptor is not linked to PKCϵ phosphorylation. A similar PKCϵ response was observed for primary human macrophages. In freshly isolated macrophages challenged with LPS, LTA, poly(I-C), or flagellin, an increase in PKCϵ phosphorylation at both Ser-346 and Ser-368 was observed with all ligands except poly(I-C) (TLR3) as indicated by PKCϵ binding to GST-14-3-3β (Fig. 3E). The induction observed for LTA was ∼2-fold compared with the 4–6-fold induction for LPS and flagellin. As noted above these responses are indicative of ligand-induced PKCϵ phosphorylation at 346/368 sites, although we cannot distinguish the changes in the individual sites by this procedure. MyD88 Links PKCϵ to TLRs—The pattern of PKCϵ responses to these TLRs parallels their engagement of MyD88, i.e. all but TLR3. To test whether MyD88 was responsible for linking PKCϵ to TLRs, cells from MyD88 knock-out mice were tested for responses to LPS using capture on GST-14-3-3β beads to monitor endogenous PKCϵ Ser-346/Ser-368 phosphorylation as evidenced by the increased recovery of PKCϵ complexed to 14-3-3β. By contrast to MyD88-replete MEFs, no response to LPS was observed in MyD88 knock-out cells (Fig. 4A). Peritoneally elicited macrophages from WT mice also responded to LPS with a substantial increase in PKCϵ recovered in a 14-3-3β pulldown, equivalent to that observed with the potent combination of TPA/calyculin. By contrast there was no LPS-induced recovery of PKCϵ from the MyD88 knock-out macrophages, despite a “WT” response to TPA/calyculin (Fig. 4B). The evidence from the knock-out model demonstrates a requirement for MyD88 in the TLR4-triggered PKCϵ response. To confirm this in an acute model, siRNAs to MyD88 were employed to knock down MyD88 expression. As illustrated in Fig. 4C, siRNA knockdown of MyD88 also abrogated the LPS-induced PKCϵ Ser-346 phosphorylation. The specificity of the effect of MyD88 knockdown on Ser-346 phosphorylation was confirmed by rescue experiments with mouse MyD88. As shown in Fig. 4D, re-expression of mouse MyD88 in cells with knockdown of endogenous MyD88 by human-specific siRNA rescued the Ser-346 phosphorylation. Interestingly, when FLAG-tagged MyD88 or untagged MyD88 is overexpressed in 293/hTLR4 cells, constitutively high Ser-346 phosphorylation is observed with no further increase in LPS stimulation (Figs. 4, E and F). This reflects elevated p38 phosphorylation in these MyD88 overexpressing cells (data not shown). PKCϵ Is Complexed with TLR4 via MyD88—The adaptor role of MyD88 and its requirement for linking PKCϵ to TLR4 suggested that PKCϵ may be physically associated with the (active) receptor. By employing FLAG-tagged TLR4, it was found that a fraction of co-expressed GFP-PKCϵ or myc-PKCϵ (Fig. 5A) could be recovered in TLR4 immunoprecipitates in an LPS-inducible manner; much lower levels of PKCϵ were recovered in anti-FLAG control immunoprecipitates (data not shown and see Fig. 5B). The complex form
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