Related Experiment Video

Updated: Aug 13, 2026

Purification of Ubiquitinated p53 Proteins from Mammalian Cells
10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

Ubiquitination of p53 at multiple sites in the DNA-binding domain

Wan Mui Chan1, Man Chi Mak, Tsz Kan Fung

  • 1Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

Molecular Cancer Research : MCR
|February 1, 2006
PubMed

Insights

The tumor suppressor p53 is ubiquitinated by MDM2. Unexpectedly, ubiquitination occurs in the DNA-binding domain, not the COOH-terminus, impacting p53 stability.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Protein Biochemistry

Background:

  • The tumor suppressor p53 is a critical regulator of cellular responses to stress.
  • p53 function is tightly controlled by negative regulators, including the ubiquitin ligase MDM2.
  • MDM2 targets p53 for degradation via ubiquitination, primarily at the N-terminus.

Purpose of the Study:

  • To precisely identify the ubiquitination sites of p53 mediated by MDM2.
  • To investigate the role of different p53 domains in MDM2-dependent ubiquitination.
  • To understand how ubiquitination affects p53 stability and function.

Main Methods:

  • Site-directed mutagenesis of p53 lysine residues.
  • In vitro ubiquitination assays using recombinant MDM2 and p53.
  • Proteolytic cleavage mapping to identify ubiquitination sites.
  • Analysis of p53 stability upon domain deletion.

Main Results:

  • Substitution of COOH-terminal lysine residues did not prevent MDM2-mediated ubiquitination.
  • p53 ubiquitination was found to occur in the NH2-terminal portion, specifically within the DNA-binding domain.
  • Conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination.
  • Deletion of the DNA-binding domain reduced ubiquitination and increased p53 stability.

Conclusions:

  • p53 ubiquitination by MDM2 is not restricted to the COOH-terminal region as previously suggested.
  • The DNA-binding domain of p53 contains critical ubiquitination sites essential for MDM2-mediated regulation.
  • These findings reveal novel regulatory mechanisms controlling p53 stability and function.

More Related Videos

Yeast As a Chassis for Developing Functional Assays to Study Human P5314:57

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Published on: August 4, 2019

Detection of Protein Ubiquitination09:00

Detection of Protein Ubiquitination

Published on: August 19, 2009

Related Concept Videos

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.
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 daughter...
DNA Damage can Stall the Cell Cycle02:36

DNA Damage can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
DNA Damage Can Stall the Cell Cycle02:36

DNA Damage Can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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...
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.