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

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.