The kinase activity of Rip1 is not required for tumor necrosis factor-alpha-induced IkappaB kinase or p38 MAP kinase activation or for the ubiquitination of Rip1 by Traf2.
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.
Lee
Thomas H
TH
Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Shank
Jennifer
J
Cusson
Nicole
N
Kelliher
Michelle A
MA
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D004622:Embryo, Mammalian; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts; D020690:GTPase-Activating Proteins / Q000172:deficiency / Q000502:physiology*; D051550:I-kappa B Kinase; D015850:Interleukin-6 / Q000096:biosynthesis; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I; D047992:TNF Receptor-Associated Factor 2; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases; D000099151:MAP Kinase Kinase Kinase 7
D015703:Antigens, CD; D020690:GTPase-Activating Proteins; D051550:I-kappa B Kinase; D015850:Interleukin-6; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; D048051:p38 Mitogen-Activated Protein Kinases; D000099151:MAP Kinase Kinase Kinase 7; C490620:Ralbp1 protein, mouse; C496564:Chuk protein, mouse; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse