Inhibiting TAK1 Serine 412 Phosphorylation Negatively Regulates TLR Response by Phosphatase Holoenzyme PP1/GADD34

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-kB pathway and the production of TNF-a, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-b–activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R–triggered NF-kB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflamma-tory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphoryla-tion, downstream NF-kB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses. T oll-like receptors play critical roles in innate immunity and host defense. They detect invading pathogens through recognition of pathogen-associated molecular patterns (PAMPs) such as LPSs, nucleic acids, lipoproteins, and glycans derived from pathogens and initiate the activation of multiple intracellular signaling pathways (1). Among these pathways, NF-kB and MAPK pathways are most important (2). Activation of the NF-kB and MAPK pathways leads to induction of cytokines and chemokines, which shape the nature, magnitude, and duration of host proinflammatory and innate immune responses (3). Although full activation of TLR signaling is essential for host defense, dysregulation of this process contributes to many inflammatory diseases and immunopathologic disorders, such as endotoxin shock and autoimmune disease (2). It is important to elucidate the underlying molecular mechanism through which the TLR response is tightly regulated. For example, previous studies identified positive regulators of the TLR pathway, such as the, Peli1, Nrdp1, and MHC class II molecules (4–6), and negative regulators such as NLRC3, SHP-2, and MHC …