Novel Regulation Of Protein Kinase TAK1Activation By Protein Kinase A And Salicylates

TGF-β-activated kinase1(TAKl) is a key kinase in mediating NF-κB activation, including Toll-like receptors (TLRs) and interleukin-1receptor (IL-1R) signaling. Although TAK1activation involves the phosphorylation of Thr184and Thr187residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1remains elusive. In this work, we show that the Thrl87phosphorylation of TAK1is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1activation in mediating downstream signaling requires an additional phosphorylation at Ser412, which is critical for TAK1response to proinflammatory stimuli, such as TNF-a, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1Ser412phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit a (PKACa) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser391in zebrafish TAKl plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1phosphorylation mediated by PKACa and PRKX that contributes to innate immune signaling. Salicylic acid and its derivatives are widely-used nonsteroidal anti-inflammatory drugs (NSAIDs). Besides their anti-inflammatory and analgesic properties, they can also prevent cardiovascular diseases and even some type of cancer. All these effects are partly ascribed to the inhibition of NF-κB activation. Although it was found decade ago that salicylates directly inhibit the activity of IKKβ due to binding of these agents to IKKβ to reduce ATP binding, the exact mechanism of their anti-inflammation effect remains elusive. Here we show that upon TNF-a or IL-1β stimulation the activation of TAK1, the upstream kinase of IKKs, is blocked by sodium salicylate and aspirin. Further in vitro study show that salicylates do not inhibit the kinase activity of TAK1and IKKβ per se. In fact, salicylates repress TAK1activation by inhibiting stimulation induced ubiquitination in vivo. Moreover, salicylates directly inhibit the synthesis of K63-linked and Met1-linked poly-ubiquitin chain in vitro. Finally, high-dose salicylates are able to alleviate lipopolysaccharide-induced acute lung injury in mice, partly due to inhibition of NF-κB activation and the assembly of TNF receptor signaling complex in lung tissues. Collectively, our data unravel a previously unknown mechanism that salicylates target the ubiquitination system to prevent activation of TAK1and its downstream signaling. This study also provides new insights into the pharmacological actions of aspirin and salicylate preparations and implicates the ubiquitination system as potential target for anti-inflammatory drug design.