Related Experiment Video

Updated: Aug 7, 2026

Purification of Ubiquitinated p53 Proteins from Mammalian Cells
10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

L2DTL/CDT2 and PCNA interact with p53 and regulate p53 polyubiquitination and protein stability through MDM2 and

Damon Banks1, Min Wu, Leigh Ann Higa

  • 1Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

Cell Cycle (Georgetown, Tex.)
|July 25, 2006
PubMed

Insights

The CUL4-ROC1 E3 ligase complex, with L2DTL and PCNA, controls p53 and MDM2/HDM2 stability. DNA damage regulates these interactions, impacting cell cycle progression and genome stability.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The CUL4-ROC1 E3 ligase complex is crucial for regulating genome stability, DNA replication, and cell cycle progression.
  • Understanding the regulatory mechanisms of key proteins like p53 and MDM2/HDM2 is vital for comprehending cellular responses to stress and maintaining genomic integrity.

Purpose of the Study:

  • To investigate the role of novel WD40 domain-containing protein L2DTL/CDT2 and PCNA in association with CUL4/DDB1 complexes.
  • To elucidate the function of the DDB1-CUL4A complex in regulating the stability of p53 and its inhibitor MDM2/HDM2.
  • To determine how DNA damage influences the interaction between p53 and the CUL4 complex.

Main Methods:

  • Protein complex isolation and identification.
  • Co-immunoprecipitation assays to study protein interactions.
  • Ubiquitination assays to assess protein degradation.
  • Western blotting to detect protein stabilization and degradation.

Main Results:

  • L2DTL/CDT2 and PCNA associate with CUL4/DDB1 complexes, forming functional units that regulate p53 and MDM2/HDM2 stability.
  • Inactivation of components like CUL4A, L2DTL, PCNA, DDB1, or ROC1 leads to p53 stabilization and cell growth arrest.
  • The DDB1-CUL4A complex exhibits robust polyubiquitination activity towards p53, dependent on L2DTL, PCNA, and MDM2/HDM2.
  • MDM2/HDM2 undergoes rapid proteolysis upon UV irradiation, a process mediated by the CUL4/DDB1 and PCNA complex.
  • The interaction between p53 and the CUL4 complex is modulated by DNA damage.

Conclusions:

  • PCNA, L2DTL, and the DDB1-CUL4A complex play critical, distinct roles in controlling p53 and MDM2/HDM2 protein stability under both normal and stressed conditions.
  • These findings highlight a novel regulatory pathway involving the CUL4-ROC1 E3 ligase complex in maintaining genome stability and cell cycle control.
  • The study provides insights into the dynamic regulation of tumor suppressor p53 and its regulator MDM2/HDM2 in response to cellular stress.

More Related Videos

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications10:12

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Published on: April 21, 2023

Detection of Protein Ubiquitination09:00

Detection of Protein Ubiquitination

Published on: August 19, 2009

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 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...
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.
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.
Inhibition of Cdk Activity02:34

Inhibition of Cdk Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...