Polyubiquitination of Transforming Growth Factor β (TGFβ)-associated Kinase 1 Mediates Nuclear Factor-κB Activation in Response to Different Inflammatory Stimuli

The transcription factor nuclear factor kappa B (NF-kappa B) plays a central role in regulating inflammation in response to several external signals. The TGF beta-associated kinase 1 (TAK1) is an upstream regulator of NF-kappa B signaling. In TGF beta-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-kappa B activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1 beta. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-alpha and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGF beta, which in all cases resulted in NF-kappa B activation. Expression of a K34R-mutant TAK1 led to a reduced NF-kappa B activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-alpha-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-kappa B signaling downstream of several receptors.