title: The scaffold MyD88 acts to couple protein kinase Cepsilon to Toll-like receptors.
abstract: Mice lacking protein kinase Cepsilon (PKCepsilon) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCepsilon coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCepsilon involvement in TLR signaling and found that PKCepsilon 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 PKCepsilon binding to 14-3-3beta. LPS-induced PKCepsilon phosphorylation, 14-3-3beta 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 PKCepsilon 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/PKCepsilon association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCepsilon. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCepsilon 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 PKCepsilon at these two sites is required for TLR4- and TLR2-induced NFkappaB reporter activation and IkappaB degradation in reconstituted PKCepsilon(-/-) cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCepsilon recruitment, phosphorylation, and downstream signaling.
authors.affiliation: Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, London WC2A 3PX, United Kingdom.
authors.forename: Amir
authors.initials: A
authors.lastname: Faisal
authors.forename: Adrian
authors.initials: A
authors.lastname: Saurin
authors.forename: Bernard
authors.initials: B
authors.lastname: Gregory
authors.forename: Brian
authors.initials: B
authors.lastname: Foxwell
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Parker
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian / Q000276:immunology / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects / Q000276:immunology; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D016905:Gram-Negative Bacterial Infections / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016908:Gram-Positive Bacterial Infections / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects / Q000502:physiology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; C482982:YWHAB protein, human; D051744:Protein Kinase C-epsilon