Related Experiment Video

Updated: Sep 2, 2025

Analysis of Cell Cycle Position in Mammalian Cells
12:19

Analysis of Cell Cycle Position in Mammalian Cells

Published on: January 21, 2012

60.6K

Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function

Dominika Kowalczyk1, Mark A Nakasone1, Brian O Smith2

  • 1Cancer Research UK Beatson Institute, Glasgow, UK.

Life Science Alliance
|August 9, 2022
PubMed

Insights

The ARF tumor suppressor protein inhibits MDM2 E3 ligase activity by binding to both the acidic and RING domains of MDM2. This dual binding mechanism blocks E2∼ubiquitin recruitment, revealing how ARF regulates the MDM2-p53 pathway.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cancer Research

Background:

  • ARF (alternative reading frame) is a crucial tumor suppressor.
  • It regulates the MDM2-p53 signaling axis, impacting cell growth and survival.
  • ARF inhibits MDM2's ubiquitin ligase activity, but the precise mechanism was unclear.

Purpose of the Study:

  • To elucidate the molecular mechanism of ARF-mediated inhibition of MDM2 ubiquitin ligase (E3) activity.
  • To investigate the structural basis of ARF-MDM2 interaction and its effect on E3 function.

Main Methods:

  • Nuclear magnetic resonance (NMR) spectroscopy.
  • Biochemical assays to analyze protein interactions and enzyme activity.

Main Results:

  • MDM2's acidic and zinc finger domains (AD-ZnF) weakly interact with its RING domain, partially inhibiting E3 activity.
  • p14ARF binding to MDM2's acidic domain enhances the AD-ZnF-RING interaction.
  • p14ARF's RxFxV motifs directly engage the MDM2 RING domain, creating a bivalent interaction.
  • This bivalent binding obstructs E2∼ubiquitin binding, significantly hindering MDM2 E3 activity.

Conclusions:

  • ARF inhibits MDM2 E3 activity through a bivalent binding mechanism involving both MDM2 acidic and RING domains.
  • This detailed mechanism clarifies how ARF functions as a tumor suppressor by modulating p53 stability.

More Related Videos

Visualization of DNA Repair Proteins Interaction by Immunofluorescence07:55

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Published on: June 26, 2020

10.4K
Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay09:07

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Published on: December 19, 2018

6.4K

Related Concept Videos

Abnormal Proliferation02:23

Abnormal Proliferation

Under normal conditions, most adult cells remain in a non-proliferative state unless stimulated by internal or external factors to replace lost cells. Abnormal cell proliferation is a condition in which the cell's growth exceeds and is uncoordinated with normal cells. In such situations, cell division persists in the same excessive manner even after cessation of the stimuli, leading to persistent tumors. The tumor arises from the damaged cells that replicate to pass the damage to the...
4.6K
Negative Regulator Molecules01:23

Negative Regulator Molecules

Positive regulators allow a cell to advance through cell cycle checkpoints. Negative regulators have an equally important role as they terminate a cell’s progression through the cell cycle—or pause it—until the cell meets specific criteria.
35.8K
Separation of Sister Chromatids02:17

Separation of Sister Chromatids

At the transition from prophase to metaphase, there is a reduction in cohesion along the chromosomal arms, resulting in the resolution of sister chromatids. However, residual cohesin connections remain to hold the sister chromatids together until the transition from metaphase to anaphase. The residual connection prevents any premature separation of sister chromatids, blocking the risks of aneuploidy within the daughter cells.
At the onset of anaphase, separase, a proteolytic enzyme, is...
3.8K
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein....
7.0K
Anaphase Promoting Complex00:50

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
2.9K
Master Transcription Regulators02:23

Master Transcription Regulators

Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a  complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
7.0K