Abstract
TAK1 (transforming growth factor-beta-activated kinase 1), a mitogen-activated protein kinase kinase kinase, is activated by various cytokines, including interleukin-1 (IL-1). However, the precise regulation for TAK1 activation at the molecular level is still not fully understood. Here we report that dual phosphorylation of Thr-178 and Thr-184 residues within the kinase activation loop of TAK1 is essential for TAK1-mediated NFkappaB and AP-1 activation. Once co-overexpressed with TAB1, TAK1 mutant with alanine substitution of these two residues fails to activate IKKbeta-mediated NFkappaB and JNK-mediated AP-1, whereas TAK1 mutant with replacement of these two sites with acidic residues acts like the TAK1 wild type. Consistently, TAK1 mutant with alanine substitution of these two residues severely inhibits IL-1-induced NFkappaB and AP-1 activities, whereas TAK1 mutant with replacement of these two sites with acidic residues slightly enhances IL-1-induced NFkappaB and AP-1 activities compared with the TAK1 wild-type. IL-1 induces the phosphorylation of endogenous TAK1 at Thr-178 and Thr-184. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with wild-type TAK1 or a TAK1 mutant containing threonine 178 and 184 to alanine mutations revealed the importance of these two sites in IL-1-mediated IKK-NFkappaB and JNK-AP-1 activation as well as IL-1-induced IL-6 gene expression. Our finding is the first report that substitution of key serine/threonine residues with acidic residues mimics the phosphorylated state of TAK1 and renders TAK1 active during its induced activation.
Highlights
TAK1, a member of the evolutionarily conserved mitogen-activated protein kinase kinase kinase family, was originally found to function in signaling of the transforming growth factor- [6]
These results strongly suggest that the TAK1 T178E/T184E mutant is active in the assays, and the phosphorylation of both Thr-178 and Thr-184 residues is required for the TAK1/TAB1-induced nuclear transcription factor B (NFB) and AP-1 activities
These results demonstrate dual phosphorylation of the Thr-178 and Thr-184 residues is that our antibody can recognize both TAK1 T178A and T184A required for TAK1/TAB1-induced Jun NH2-terminal kinase (JNK) and p38 activation, the single mutant and suggest that Ala substitution at Thr-178 had wild-type, catalytically inactive K63R and Thr-178 or/and Thrno effect on the phosphorylation of Thr-184 on the activated 184 TAK1 mutants were overexpressed in HEK-293T cells
Summary
Cell Culture and Transfection—TAK1-deficient MEF cells have been described before (40 – 42). Cells were lysed by adding lysis buffer (25 mM HEPES (pH 7.7), 135 mM NaCl, 3 mM EDTA, 1% Triton X-100, 25 mM -glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM benzamidine, 20 mM disodium p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, phosphatase inhibitor mixture 1 and 2 (Sigma)). Cytoplasmic extracts were prepared by adding Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 mM glycerophosphate, 0.1 mM sodium orthovanadate, 10 g/ml aprotinin, and 10 g/ml leupeptin) to cell pellets. Enzyme-linked Immunosorbent Assay—Supernatants of stable MEF-TAK1 knock-out cell lines expressing TAK1 wild type and TAK1-T178A/T184A, which were treated with or without mouse IL-1 (10 ng/ml), were collected at different time points. In Vitro Phosphatase Treatment—HEK293T cells were transfected with FLAG-TAK1 and TAB1 expression vectors, and cell extracts were prepared in protein lysis buffer.
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