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

Abstract

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-κB pathway and the production of TNF-α, 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-β–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-κB 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 proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB 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.

Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

“…However, some studies have shown that the phosphorylation of these sites is insufficient for TAK1 activation (14,15). Consistent with this notion, our recent investigation with PP1 implied that the phosphorylation of TAK1 Ser-412 is important for TAK1 activation and function (30).…”

“…In summary, the present study together with our recent report (30) showing a negative regulation of TAK1 by the phosphatase PP1-GADD34 complex provided strong evidence that TAK1 activation required the phosphorylation of TAK1 Ser-412 in the IL-1R and TLR signaling pathways. PKA kinases, by directly phosphorylating TAK1, were found to be important regulators of proinflammatory and innate immune responses.…”

“…We have recently demonstrated that protein phosphatase 1 (PP1), together with its adaptor GADD34, modulates IL-1R/ TLR signaling through the dephosphorylation of TAK1 at Ser-412 (30). In this report, we aimed to further study the regulation of TAK1 activation and Ser-412 phosphorylation.…”

Preclinical Assessment of Mutant Human TRIM5α as an Anti-HIV-1 Transgene

“…69,70 We used Western blotting to analyze the total and Ser412-phosphorylated forms of TAK1 in stably transduced CEM.NKR-CCR5 cells (Fig. 3A).…”

Impact of GADD34 on Apoptosis of Tonsillar Mononuclear Cells from IgA Nephropathy Patients by Regulating Eif2α Phosphorylation

“…A high expression of GADD34 can dramatically decrease the susceptibility of Oca2-null melanocytes to thapsigargin [27]. It can also prevent the cell damage after TLR stimulation and inflammation [28]. Thus, GADD34 upregulation may function as a protective mechanism in cells under ER stress.…”