Related Experiment Video

Updated: Jun 6, 2026

Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System
08:15

Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System

Published on: April 11, 2025

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling

Jasmin N Dynek1, Tatiana Goncharov, Erin C Dueber

  • 1Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.

The EMBO Journal
|November 30, 2010
PubMed

Insights

Cellular inhibitor of apoptosis proteins (c-IAP1/2) and UbcH5 enzymes mediate K11-linked polyubiquitination of RIP1, crucial for TNFα-induced NF-κB activation. This specific ubiquitin linkage may play a role in cell proliferation pathways.

Area of Science:

  • * Molecular Biology
  • * Cell Signaling
  • * Ubiquitination Pathways

Background:

  • * Ubiquitin ligases and E2 enzymes control polyubiquitin chain assembly, dictating substrate fate.
  • * Cellular inhibitors of apoptosis (c-IAP1/2) are recruited to TNFR1 complexes, regulating NF-κB activation via their RING domain ubiquitin ligase activity.

Purpose of the Study:

  • * To identify novel E2 enzyme partners for IAP RING domains.
  • * To investigate the role of c-IAP1 and UbcH5 in regulating c-IAP1 stability and NF-κB activation.
  • * To elucidate the specific type of polyubiquitin chain linkage involved in TNFR1 signaling.

Main Methods:

  • * Directed yeast two-hybrid screening to identify E2 enzyme partners.
  • * In vitro and in vivo experiments to assess polyubiquitination of RIP1.
  • * Analysis of TNFα-stimulated NF-κB activation in the context of c-IAP1 and UbcH5 function.

Main Results:

  • * Identified UbcH5 family as critical regulators of c-IAP1 stability following various stimuli.
  • * Demonstrated that c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1.
  • * Showed that TNFα-induced NF-κB activation depends on endogenous K11-linked ubiquitination of RIP1 within the TNFR1 complex, mediated by c-IAP1 and UbcH5.
  • * Found that NF-κB essential modifier binds K11-linked ubiquitin chains.

Conclusions:

  • * UbcH5 family E2 enzymes are key partners of c-IAP1, regulating its stability and function.
  • * K11-linked polyubiquitination of RIP1 by c-IAP1/UbcH5 is essential for TNFα-induced NF-κB activation.
  • * The K11-linked ubiquitin chain linkage may signal for the activation of proliferative cellular pathways.

More Related Videos

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells09:15

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Published on: October 20, 2022

In Vitro Analysis of E3 Ubiquitin Ligase Function06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Related Concept Videos

Regulation of the Unfolded Protein Response01:31

Regulation of the Unfolded Protein Response

Inositol-requiring kinase one or IRE1 is the most conserved eukaryotic unfolded protein response (UPR) receptor. It is a type I transmembrane protein kinase receptor with a distinctive site-specific RNase activity. As the binding mechanics of the misfolded proteins with the N-terminal domain of IRE-1 are unclear, three binding models — direct, indirect, and allosteric -- are proposed for receptor activation. Nevertheless, it is known that once a misfolded protein associates with IRE1, it...
The Unfolded Protein Response01:37

The Unfolded Protein Response

The ER is the hub of protein synthesis in a cell. It has robust systems to quality control protein folding and also for degradation of terminally misfolded proteins. Under normal conditions, a small proportion of misfolded proteins that cannot be salvaged need to be transported to the cytoplasm by the ER-associated degradation or ERAD pathways. However, if the ERAD cannot handle the misfolded proteins, the cell activates the unfolded protein response or UPR to adjust the protein folding...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
NF-κB-dependent Signaling Pathway02:26

NF-κB-dependent Signaling Pathway

The transcription factor NF-κB was discovered in 1986 in the lab of Nobel laureate Professor David Baltimore, for its interaction with the immunoglobulin light chain enhancer in B-cells. After more than three decades of study, it is now evident that NF-κB regulates the expression of over 100 genes. Most of these genes play an essential role in the innate and adaptive immune responses as well as the inflammatory responses of animals.
NF-κB-dependent Signaling Mechanism
The heterodimer of NF-κB...
Receptor Downregulation in MVBs01:15

Receptor Downregulation in MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
The EGFR can initiate signaling pathways that  lead to cell proliferation, migration, and differentiation. Overexpression of EGFR  stimulates cells to proliferate. Excessive  EGFR activation may...
Regulated Protein Degradation02:58

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...