title: The role of TBK1 and IKKε in the expression and activation of Pellino 1.
abstract: Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKε. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)-/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor-/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.
authors.affiliation: MRC Protein Phosphorylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Hilary
authors.initials: H
authors.lastname: Smith
authors.forename: Xin-Yu
authors.initials: XY
authors.lastname: Liu
authors.forename: Liang
authors.initials: L
authors.lastname: Dai
authors.forename: Eddy T H
authors.initials: ET
authors.lastname: Goh
authors.forename: Aye-Thu
authors.initials: AT
authors.lastname: Chan
authors.forename: Jiajia
authors.initials: J
authors.lastname: Xi
authors.forename: Cheah-Chen
authors.initials: CC
authors.lastname: Seh
authors.forename: Insaf A
authors.initials: IA
authors.lastname: Qureshi
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.forename: Christiane
authors.initials: C
authors.lastname: Ruedl
authors.forename: Robert
authors.initials: R
authors.lastname: Gourlay
authors.forename: Simon
authors.initials: S
authors.lastname: Morton
authors.forename: Joanne
authors.initials: J
authors.lastname: Hough
authors.forename: Edward G
authors.initials: EG
authors.lastname: McIver
authors.forename: Philip
authors.initials: P
authors.lastname: Cohen
authors.forename: Peter C F
authors.initials: PC
authors.lastname: Cheung
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase / Q000502:physiology*; D050838:Interferon Regulatory Factor-3 / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics; D009687:Nuclear Proteins / Q000096:biosynthesis*; D010766:Phosphorylation; D011070:Poly I-C / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D053633:Receptor, Interferon alpha-beta / Q000502:physiology; D015398:Signal Transduction; D051196:Toll-Like Receptor 3 / Q000819:agonists; D051197:Toll-Like Receptor 4 / Q000819:agonists; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D051550:I-kappa B Kinase; D050838:Interferon Regulatory Factor-3; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D009687:Nuclear Proteins; D011070:Poly I-C; D017346:Protein Serine-Threonine Kinases; D053633:Receptor, Interferon alpha-beta; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D044767:Ubiquitin-Protein Ligases; C494233:Irf3 protein, mouse; C507780:Myd88 protein, mouse; C485961:Peli2 protein, mouse; C476286:Ticam1 protein, mouse; C519703:TLR3 protein, mouse; C493487:Tlr4 protein, mouse; C498588:Tbk1 protein, mouse; C418046:Peli1 protein, mouse