The Kinase Activity of Rip1 Is Not Required for Tumor Necrosis Factor-α-induced IκB Kinase or p38 MAP Kinase Activation or for the Ubiquitination of Rip1 by Traf2

Michelle A Kelliher,Nicole Cusson,Jennifer Shank,Thomas H Lee

doi:10.1074/jbc.m404206200

Michelle A Kelliher, Nicole Cusson + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m404206200

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Abstract

The death domain kinase Rip1 is recruited to the tumor necrosis factor receptor type 1 and mediates the IkappaB kinase and p38 MAP kinase pathways. In response to tumor necrosis factor-alpha (TNF-alpha), we find Rip1 phosphorylated and ubiquitinated, suggesting that Rip1 phosphorylation may stimulate its ubiquitination. To address the contribution of the kinase activity of Rip1 to its ubiquitination and to TNF-alpha signaling, we introduced wild type Rip1 and a kinase-inactive form of Rip1, Rip1D138N, into rip1-/- murine embryonic fibroblast cells by retroviral infection. TNF-alpha-induced ubiquitination of Rip1 is observed in Rip1D138N cells, supporting the argument that Rip1 autophosphorylation is not required for Rip1 ubiquitination. TNF-alpha-induced Ikk and p38 MAP kinase activation is normal, and the Rip1D138N cells are resistant to TNF-alpha-induced cell death, indicating that the kinase activity of Rip1 is not required to mediate its antiapoptotic functions. In the absence of Traf2, TNF-alpha-induced ubiquitination of Rip1 is impaired, suggesting that Traf2 may be the E3 ubiquitin ligase responsible for the TNF-alpha-dependent, ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in signaling.

Highlights

The proinflammatory cytokine tumor necrosis factor-␣ (TNF␣)1 is a major mediator of apoptosis as well as inflammation and immunity
To determine whether Rip1 autophosphorylation triggers its ubiquitination and to test whether Rip1 ubiquitination contributes to MAP kinase activation, we introduced wild type rip1 and a kinase-inactive rip1 into rip1-deficient murine embryonic fibroblasts (MEFs) and examined TNF-␣-induced ubiquitination of Rip1 as well as activation of Ikk and p38 MAP kinase
We have previously demonstrated that TNF-␣-induced Ikk and p38 MAP kinase activation are both impaired in rip1-deficient MEFs [12, 14]

Summary

Introduction

The proinflammatory cytokine tumor necrosis factor-␣ (TNF␣)1 is a major mediator of apoptosis as well as inflammation and immunity. TNF-␣-induced activation of Ikk and p38 MAP is not affected in cells expressing an inactive Rip1 kinase, demonstrating that Rip1 autophosphorylation is not required for signaling. To determine whether Rip1 ubiquitination occurs at the Tnfr1, wild type cells or cells expressing kinase-inactive Rip1 were left untreated or treated with mTNF-␣ for the time periods indicated.

Results

Conclusion