Regulation of TLR4 signaling through the TRAF6/sNASP axis by reversible phosphorylation mediated by CK2 and PP4

Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor-associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor-associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-alpha production. Survival of Ad-PP4-infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.