Related Experiment Video

Updated: Jun 26, 2026

Detection of Protein Ubiquitination
09:00

Detection of Protein Ubiquitination

Published on: August 19, 2009

Down-regulation of active ACK1 is mediated by association with the E3 ubiquitin ligase Nedd4-2

Wing Chan1, Rui Tian, Yeow-Fong Lee

  • 1GSK, Institute of Molecular and Cell Biology, Singapore 138673.

The Journal of Biological Chemistry
|January 16, 2009
PubMed

Insights

Activated Cdc42-associated kinase 1 (ACK1) is degraded by the E3 ubiquitin ligase Nedd4-2, a process regulated by epidermal growth factor (EGF) signaling and impacting cell receptor dynamics.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Activated Cdc42-associated kinase 1 (ACK1) is a cytoplasmic tyrosine kinase involved in epidermal growth factor (EGF) receptor and clathrin-mediated trafficking.
  • Understanding the regulation of ACK1 levels is crucial for comprehending cell receptor dynamics.

Purpose of the Study:

  • To identify novel binding partners of ACK1.
  • To elucidate the regulatory mechanisms controlling ACK1 protein levels.
  • To investigate the role of ACK1 regulation in cellular signaling pathways.

Main Methods:

  • Co-immunoprecipitation assays to identify ACK1-binding proteins.
  • Western blotting to assess protein levels and ubiquitination.
  • Confocal microscopy to determine subcellular localization.
  • Treatment with proteasome inhibitors (e.g., MG132) to study protein degradation pathways.

Main Results:

  • Nedd4-2, an E3 ubiquitin ligase, was identified as a novel binding partner of ACK1, interacting via a conserved PPXY motif.
  • Nedd4-2 mediates the down-regulation of ACK1 protein levels through proteasomal degradation.
  • ACK1 degradation is dependent on Nedd4-2 interaction, E3 ligase activity, and ACK1 kinase activity.
  • Epidermal growth factor (EGF) stimulation leads to ACK1 activation, which subsequently promotes Nedd4-2-mediated ACK1 degradation.
  • ACK1 undergoes polyubiquitination in vivo, and Nedd4-2 activity is essential for its turnover following EGF treatment.

Conclusions:

  • The E3 ubiquitin ligase Nedd4-2 plays a critical role in regulating ACK1 protein stability.
  • EGF receptor-mediated activation of ACK1 facilitates its degradation by Nedd4-2, linking kinase activation to protein turnover.
  • The interplay between Nedd4-2, ACK1, and other regulatory proteins like Cbl impacts EGF receptor dynamics.
  • Dysregulation of ACK1, potentially through genomic amplification in metastatic tumors, highlights the clinical relevance of these regulatory mechanisms.

More Related Videos

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale10:50

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Published on: March 14, 2019

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Related Concept Videos

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...
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...
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...
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...
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...
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.