Regulation Of TNF-α And IL-1β-induced NF-κB Signaling Pathway By DUSP14

The transcription factor NF-κB(Nuclear factor-kappaB) which exist in many kinds of cell is a very significant nuclear transcription factor. NF-κB plays an important role in the regulation of many immune and inflammatory responses. NF-κB is involved in the regulation of the expression of a variety of proteins in cell.Inside the cell, protein phosphorylation is a reversible dynamic regulation process of protein modification. Protein kinase and phosphatase have different function in the regulation of the level of protein phosphorylation. Protein phosphorylation and dephosphorylation regulated by its kinase and phosphatase can ensure that the level of protein phoaphorylation is maintained in a dynamic balance state.Reversible protein phosphorylation plays a very significant role in the TNFa-or IL-1β-triggered NF-κB signaling pathways. For examples, the phosphorylation of TAK1, IKK or p65plays an important role in NF-κB pathways. We speculate that several new protein kinases or protein phosphatases maybe involved in the TNFa-or IL-1β-triggered NF-κB signaling pathways.To identify some additional proteins which regulate NF-κB activation, we screened a mouse cDNA library containing3000expression clones which purchased from Origene Inc. by performing the dual luciferase reporter assays. In these experiments,3,000mouse cDNA clones were separately transfected into HEK293cells together with NF-κB luciferase reporter plasmid and TK Renilla luciferase reporter plasmid. Twenty hours after transfection, cells were left untreated or treated with TNFa or IL-1β for eight hours and then subjected to reporter assays to detect the function on the TNFa-or IL-1β-triggered NF-κB activation by these3,000proteins which translated from the3,000mouse cDNA clones.We got a protein which can strongly inhibited the TNFa-or IL-1β-induced NF-κB activation through luciferase reporter assays. This protein which name is DUSP14belongs to DUSP14(dual-specificity phosphatases) family. DUSP14is approximately198amino-acids residues in length and is composed of a single catalytic phosphatase domain (Dual-specificity phosphatase catalytic domain, DSPs). The phosphate-binding pocket of DUSP14is formed by a loop containing the active-site motif is comprised of the sequence H110CAAGVSR117, and the catalytic site is cysteine111.The results of dual luciferase reporter assay indicated that overexpression of DUSP14can reduce TNFa-or IL-1β-induced NF-κB activation, and knockdown of DUSP14can enhance the NF-κB activation induced by TNFa and IL-1β. In this study, our results suggested that DUSP14negatively regulates NF-κB signaling pathway through dephosphorylating TAK1. First, DUSP14and TAK1have interaction in overexpression condition and endogenous condition. Second, overexpression of DUSP14can strongly inhibit TNFa-or IL-1β-mediated TAK1activation, and phosphatase-deficient mutant DUSP14(C111S) can not reduce TAK1activation. Third, in overexpression system, wild type DUSP14bot not phosphatase-deficient mutant DUSP14(C111S) could dephosphorylate TAK1. In endogenous condition, knockdown od DUSP14could enhance TNFa-and IL-1β-induced phosphorylation of TAK1. So DUSP14is a TAK1phosphatase which can reduce TAK1activation.Protein phosphorylation and dephosphorylation play a very significant role in the TNFa-or IL-1β-triggered NF-κB signaling pathways. Our results indicated that phosphatase DUSP14has very important function in NF-κB signaling pathway, DUSP14could reduce TAK1activation by dephosphorylating TAK1. Our study further showed the important role of phosphrylation and dephosphorylation in NF-κB signaling pathway, and provided new evidence for further exploration of the protein phosphatase functions in the NF-κB signaling pathways.TAK1is a very important adapter protein in TNFa-and IL-1β-triggered NF-κB signaling pathways. But the regulatory mechanism of TAK1function is still not very clear. Our study found a new mechanism of TAK1modification after its translation, and provided some new evidence for further exploration of TAK1.