The Role of TAK1 Associated Binding Protein 2 in Tumor Necrosis Factor Signaling and Cell Death

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Title: The Role of TAK1 Associated Binding Protein 2 in Tumor Necrosis Factor Signaling and Cell Death
Author: Broglie, Peter Michael
Advisors: Jun Ninomiya-Tsuji, Committee Chair
James C. Bonner, Committee Member
Yoshiaki Tsuji, Committee Member
Robert C. Smart, Committee Member
Abstract: Tumor Necrosis Factor (TNF) is a key modulator of the innate immune system, aiding in the activation of immune cells as well as killing damaged cells. Dysregulation of the TNF-initiated intracellular signaling pathways can cause a number of pathologic conditions. TNF binds to TNF receptor (TNFR) and initiates signaling by assembling a protein complex on the receptor consisting of Receptor Interacting Protein 1 (RIP1) and TNF-Receptor Associated Factor 2/5 (TRAF2/5). The RIP1-TRAF complex ultimately activates the IκB kinase (IKK)-NF-κB pathway as well as mitogen activated protein kinase (MAPK)-AP-1 pathway, and transcriptionally activates various anti-inflammatory genes. A key step in this signaling pathway is the activation of the MAPK kinase kinase family member TGF-β Activated Kinase 1 (TAK1) through which the IKK and MAPK pathways are activated. Activation of TAK1 occurs within 3-5 minutes in TNF signaling and is quickly deactivated by the protein phosphatase PP6, which is important to avoid prolonged signaling. It has been demonstrated that RIP1 is ubiquitinated and this polyubiquitin chain is important for assembly of the signaling complex consisting of RIP1, TAK1, TAB2 and IKK. However, the mechanism by which PP6 is recruited to TAK1 has not yet been determined. In this study, we show that the TAK1 associated binding protein TAB2 is critical in the deactivation of TAK1 by localizing TAK1 and PP6 in close proximity. We found that, in TAB2 knockout fibroblasts, TAK1 is not deactivated in a timely manner as it is in TAB2 wild-type cells. We found that the interaction between TAK1 and PP6 is disrupted in TAB2 knockout fibroblasts. We demonstrated that PP6 was normally associated with a polyubiquitin chain, and the TAB2-polyubiquitin complex was associated with PP6. Our results have revealed that a polyubiquitin chain is important not only for the assembly of the activation complex but also of the deactivation complex for TAK1 in TNF signaling pathway, and that TAB2 participates in these complex as an indispensable adaptor. We have observed that, following prolonged exposure to TNF, TAB2 knockout fibroblasts underwent cell death. We next investigated the role of TAB2 in TNF-induced cell death. Treatment with caspase inhibitors only further sensitized cells to TNF-induced cell death, indicating that TAB2 knockout cells were not dying through an apoptotic mechanism. Treating cells with a specific necroptosis inhibitor, Necrostatin-1, we found that this cell death hypersensitivity was completely rescued, suggesting that TAB2 deficiency activates TNF-induced necroptosis. Necroptosis is an active form of necrotic cell death that is induced when apoptosis is inhibited. It has been known that a complex consist of RIP1 and a RIP1 related kinase, RIP3, plays key roles in the activation of the necroptotic pathway. However, the mechanism by which the RIP1-RIP3 is activated has not been determined. We found that TAB2 deficiency caused prolonged activation of TAK1 resulting in association of TAK1 with RIP1 in a prolonged manner. TAK1 could activate RIP1 and RIP3 kinase activity. Our results in this study demonstrate that TAB2 regulation of TAK1 is critical in the prevention of TNF-induced necroptosis. In summary, TAB2 is a dual role TAK1 adaptor protein that mediates both activation and deactivation of TAK1. The TAB2-mediated deactivation of TAK1 is particularly important, because impairment of this deactivation results in necroptosis leading to dysregulated inflammatory conditions.
Date: 2010-03-29
Degree: PhD
Discipline: Toxicology

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