Related Experiment Video

Updated: Jul 18, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination

Peter Lock1, Stacey T T I, Andrew F L Straffon

  • 1Cell Signaling Laboratory, Department of Surgery, University of Melbourne, Royal Melbourne Hospital, Parkville 3050, Australia. petelock@unimelb.edu.au

Biochemical and Biophysical Research Communications
|November 11, 2006
PubMed

Insights

Spred proteins regulate cell signaling. This study reveals Spred-2 protein ubiquitination, a key step in its degradation, is controlled by Cbl E3 ubiquitin ligases and tyrosine phosphorylation.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Signal Transduction

Background:

  • Spred proteins are negative regulators of receptor tyrosine kinase (RTK) signaling pathways, particularly the Ras-MAPK cascade.
  • Dysregulation of RTK signaling is implicated in various diseases, including cancer.

Purpose of the Study:

  • To investigate the post-translational modification and regulation of Spred proteins, focusing on ubiquitination.
  • To elucidate the role of specific tyrosine residues and E3 ubiquitin ligases in Spred-2 stability and function.

Main Methods:

  • HEK293T cells and ME4405 melanoma cells were used for experiments.
  • Techniques included stimulation with epidermal growth factor (EGF) and pervanadate, RNA interference, co-immunoprecipitation, and proteasomal inhibition (MG-132).
  • Site-directed mutagenesis was employed to identify critical tyrosine residues in Spred-2.

Main Results:

  • Spred-1, Spred-2, and Spred-3 were found to be ubiquitinated upon stimulation.
  • Spred-2 ubiquitination was dependent on specific tyrosine residues (Y228 and/or Y231) and regulated by Cbl and Cbl-b E3 ubiquitin ligases.
  • Interaction between Spred-2 and Cbl was confirmed, requiring the Cbl SH2 domain and Spred-2 Y228/Y231 residues.
  • Ubiquitination of endogenous Spred-2 led to its degradation, partially inhibited by MG-132.

Conclusions:

  • Tyrosine phosphorylation of Spred-2 at Y228/Y231 sites is critical for its ubiquitination by Cbl ligases.
  • Ubiquitination-mediated degradation plays a significant role in controlling Spred-2 protein expression levels.
  • These findings provide insights into the regulatory mechanisms of Spred proteins in growth factor signaling.

More Related Videos

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model09:00

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Published on: April 17, 2026

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)09:02

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Published on: June 10, 2020

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...
Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
Positive Regulator Molecules01:45

Positive Regulator Molecules

To consistently produce healthy cells, the cell cycle—the process that generates daughter cells—must be precisely regulated.
Positive Regulator Molecules02:39

Positive Regulator Molecules

Mitotic cell division results in daughter cells that exactly resemble the parent cell. However, errors in the DNA replication or distribution of genetic material may lead to genetic mutations that may be passed down to every new cell formed from the resulting abnormal cell. Propagation of such mutant cells is restricted through checkpoint mechanisms present at different stages of the cell cycle. These checkpoints involve regulator molecules that either promote or demote cell cycle events.
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.