The role of TBK1 and IKKε in the expression and activation of Pellino 1

Hilary Smith, Xin-Yu Liu, Liang Da, Eddy T. H. Goh, Aye-Thu Chan, Jiajia Xi, Cheah-Chen Seh, Insaf A. Qureshi, Julien Lescar, Christiane Ruedl, Robert Gourlay, Simon Morton, Joanne Hough, Edward G. McIver, Philip Cohen, Peter C. F. Cheung

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    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 I and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(l:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-beta]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [I kappa B (inhibitor of nuclear factor kappa B) kinase]related protein kinases, TBKI {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor kappa B activator]-binding kinase 1} and IKK epsilon. Pellino I 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 1KK epsilon as bait, and show that IKK epsilon/TBK1 activate Pellino 1 in vitro by phosphorylating Ser(76), Thr(288) and Ser(293). 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 IKK epsilon/TBKI and is reversed by phosphatase treatment. Thus IKK epsilon/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.

    Original languageEnglish
    Pages (from-to)537-548
    Number of pages12
    JournalBiochemical Journal
    Volume434
    DOIs
    Publication statusPublished - 15 Mar 2011

    Keywords

    • E3 ligase
    • interferon regulatory factor 3 (IRF3)
    • interleukin receptor-associated kinase (IRAK)
    • Toll-like receptor (TLR)
    • Toll/IL-1 receptor-domain-containing adaptor-inducing interferon-beta (TRIF)
    • ubiquitin
    • KAPPA-B ACTIVATION
    • E3 UBIQUITIN LIGASE
    • INTERLEUKIN-1 RECEPTOR
    • LYS63-LINKED POLYUBIQUITINATION
    • CRYSTAL-STRUCTURES
    • SIGNALING PATHWAY
    • MAMMALIAN-CELLS
    • VIRAL-INFECTION
    • INNATE IMMUNITY
    • KINASE

    Cite this

    Smith, H., Liu, X-Y., Da, L., Goh, E. T. H., Chan, A-T., Xi, J., Seh, C-C., Qureshi, I. A., Lescar, J., Ruedl, C., Gourlay, R., Morton, S., Hough, J., McIver, E. G., Cohen, P., & Cheung, P. C. F. (2011). The role of TBK1 and IKKε in the expression and activation of Pellino 1. Biochemical Journal, 434, 537-548. https://doi.org/10.1042/BJ20101421