MRC Protein Phosphorylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Biochem J. 2011 Mar 15;434(3):537-48. doi: 10.1042/BJ20101421.
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.
哺乳动物 Pellino 异构体在体外可被 IRAK（白细胞介素受体相关激酶）1/IRAK4 磷酸化，将其转化为活性 E3 泛素连接酶。在本文中，我们报道了当小鼠 BMDMs（骨髓来源的巨噬细胞）受到 LPS（脂多糖）或 poly(I:C)刺激时，Pellino 1 基因的转录和 Pellino 1 蛋白表达显著增强。这种诱导是通过 TRIF（TIR（Toll/IL-1 受体）域包含衔接诱导 IFN-β）依赖性 IRAK 非依赖性途径发生的，并且可以通过抑制 IKK（NF-κB 激酶相关蛋白激酶）、TBK1（TANK [TRAF（肿瘤坏死因子受体相关因子）相关 NF-κB 激活剂]-结合激酶 1]）和 IKKε 来阻止。IRF3（干扰素调节因子 3）缺陷型 BMDMs 中不会诱导 Pellino 1，而在 1 型干扰素受体缺陷型 BMDMs 中其诱导作用仅略有降低，这表明 Pellino 1 是一种新的 IRF3 依赖性基因。我们还使用 IKKε 作为诱饵在双杂交筛选中鉴定出 Pellino 1，并表明 IKKε/TBK1 通过磷酸化 Ser76、Thr288 和 Ser293 在体外激活 Pellino 1。此外，我们表明内源性 Pellino 1 的 E3 连接酶活性在 LPS 或 poly(I:C)刺激的巨噬细胞中被激活。这种激活发生在 Pellino 1 mRNA 和蛋白表达增加之前，可通过抑制 IKKε/TBK1 来阻止，并且可通过磷酸酶处理来逆转。因此，IKKε/TBK1 介导 Pellino 1 的 E3 连接酶活性的激活，以及 TLR3 和 TLR4 激动剂诱导其基因和蛋白表达。
