Related Experiment Video

Updated: Jun 19, 2026

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
10:27

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

HECT domain-containing E3 ubiquitin ligase Nedd4 interacts with and ubiquitinates Sprouty2

Francis Edwin1, Kimberly Anderson, Tarun B Patel

  • 1Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153, USA.

The Journal of Biological Chemistry
|October 30, 2009
PubMed

Insights

The E3 ubiquitin ligase Nedd4 targets Sprouty2 (Spry2) for degradation, regulating receptor tyrosine kinase signaling. This discovery reveals a new mechanism controlling Spry protein levels and their role in cellular signaling pathways.

Area of Science:

  • Molecular Biology
  • Cellular Signaling
  • Biochemistry

Background:

  • Sprouty (Spry) proteins are key regulators of receptor tyrosine kinase (RTK) signaling.
  • Dysregulation of Spry protein levels is implicated in cancer and cardiovascular diseases.
  • Understanding Spry regulation is crucial for therapeutic development.

Purpose of the Study:

  • To investigate the mechanisms controlling cellular Spry protein levels.
  • To identify E3 ubiquitin ligases that interact with Spry proteins.
  • To elucidate the role of Nedd4 in Spry2 stability and signaling modulation.

Main Methods:

  • Co-immunoprecipitation assays to detect Spry-Nedd4 interactions.
  • Site-directed mutagenesis to identify critical phosphorylation sites on Spry2.
  • Ubiquitination assays to assess Spry2 stability.
  • RNA interference (siRNA) to silence Nedd4 and Mnk2 expression.
  • ERK1/2 phosphorylation assays to measure RTK signaling.

Main Results:

  • Spry1 and Spry2, but not Spry3 or Spry4, associate with the E3 ubiquitin ligase Nedd4.
  • Nedd4 binding to Spry2 requires phosphorylation at Ser112/Ser121, mediated by Mnk2 kinase.
  • Nedd4 polyubiquitinates Spry2, leading to decreased protein stability.
  • Nedd4 silencing increases Spry2 levels and attenuates FGF-induced ERK1/2 activation.
  • Mnk2 silencing reduces Spry2-Nedd4 interaction and enhances Spry2's inhibitory effect on FGF signaling.

Conclusions:

  • Nedd4-mediated ubiquitination is a novel mechanism for regulating cellular Spry2 levels.
  • Phosphorylation of Spry2 by Mnk2 creates a binding site for Nedd4's WW domains.
  • This regulatory axis impacts RTK signaling, offering potential therapeutic targets.
  • This study identifies a HECT domain-containing E3 ubiquitin ligase as a key modulator of Spry-mediated signaling.

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 Analysis of E3 Ubiquitin Ligase Function06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Related Concept Videos

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...
Hedgehog Signaling Pathway02:33

Hedgehog Signaling Pathway

The Hedgehog gene (Hh) was first discovered due to its control of the growth of disorganized, hair-like bristles phenotype in Drosophila, much like hedgehog spines. Hh plays a crucial role in the development of organs and the maintenance of homeostasis in both invertebrates and vertebrates. However, while Drosophila has only one Hh protein, mammals have multiple functional Hedgehog proteins - Sonic (Shh), Desert (Dhh), and Indian Hedgehog (Ihh). All of these homologous proteins have adapted to...
Hedgehog Signaling Pathway02:33

Hedgehog Signaling Pathway

The Hedgehog gene (Hh) was first discovered due to its control of the growth of disorganized, hair-like bristles phenotype in Drosophila, much like hedgehog spines. Hh plays a crucial role in the development of organs and the maintenance of homeostasis in both invertebrates and vertebrates. However, while Drosophila has only one Hh protein, mammals have multiple functional Hedgehog proteins - Sonic (Shh), Desert (Dhh), and Indian Hedgehog (Ihh). All of these homologous proteins have adapted to...
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.
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.
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...