CpG‐DNA and its related synthetic CpG oligodeoxynucleotides (CpG‐ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG‐DNA‐responsive serine/threonine kinases; however, the target protein of CpG‐DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow‐derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) results in defective phosphorylation and activation of Akt by CpG‐DNA. Unexpectedly, loss of the Toll‐like receptor 9 has a minimal effect on Akt activation in response to CpG‐DNA. Further in vitro analysis using purified DNA‐PK and recombinant Akt proteins reveals that DNA‐PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA‐PKcs associates with Akt upon CpG‐DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA‐PKcs in CpG‐DNA signaling and define a CpG‐DNA/DNA‐PKcs/Akt pathway.

Thymosin α1 (Tα1) is noted for its immunomodulatory activities and therapeutic potential in treatment of infectious diseases and cancer. However, the molecular mechanism of its effectiveness is not completely understood. Here, we report that Tα1 induces interleukin (IL)‐6 expression through the IκB kinase (IKK) and nuclear factor‐κB (NF‐κB) pathway. Using IKKβ‐deficient bone‐marrow‐derived macrophages and mouse embryo fibroblasts (MEFs), we show that IKKβ is essential for IKK and NF‐κB activation as well as efficient IL‐6 induction. Further analysis using tumour necrosis factor receptor‐associated factor 6 (TRAF6)‐deficient MEFs shows that TRAF6 is crucial for activation of IKK and induction of IL‐6 by Tα1. Intriguingly, Tα1 triggers protein kinase C (PKC)ι/ζ activation, which is TRAF6 dependent and involves IKK. In addition, Tα1 induces the formation of a signalsome composed of TRAF6, p62 and PKCι/ζ as well as IKK. Thus, our study identifies Tα1 as a unique activator of the TRAF6 signal pathway and provides a cohesive interpretation of the molecular basis of the therapeutic utility of Tα1.

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