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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples
09:45

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Published on: May 10, 2015

Deubiquitinase activities required for hepatocyte growth factor-induced scattering of epithelial cells

Richard Buus1, Monica Faronato, Dean E Hammond

  • 1Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.

Current Biology : CB
|August 25, 2009
PubMed

Insights

Deubiquitinating enzymes (DUBs) are crucial for the invasive growth program in epithelial cells. This study identified 12 DUBs essential for Met receptor signaling and cell scattering, suggesting therapeutic targets for metastasis.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The Met receptor tyrosine kinase mediates epithelial cell scattering, a key aspect of invasive growth.
  • Ubiquitination, a posttranslational modification, regulates protein function and signaling pathways.
  • Deubiquitinating enzymes (DUBs) counteract ubiquitination, playing critical roles in cellular processes.

Purpose of the Study:

  • To identify deubiquitinating enzymes (DUBs) involved in the hepatocyte growth factor (HGF)-dependent Met receptor signaling and epithelial cell scattering.
  • To elucidate the role of the ubiquitin system in Met pathway activation and downstream signaling.

Main Methods:

  • A small interfering RNA (siRNA) library screen was employed to systematically knock down DUBs in A549 cells.
  • Phenotypic analysis of cell scattering, cell-cell adhesion, and cell motility was performed.
  • Assessment of Met receptor, phosphatidylinositol 3-kinase, and MAP kinase activation was conducted.

Main Results:

  • Twelve DUBs were identified as essential for various aspects of the HGF-dependent scattering response.
  • Specific DUBs (e.g., USP30, USP33, USP47) were critical for full scattering, while others (e.g., USP3, ATXN3L) affected cell-cell contacts and motility.
  • Knockdown of these DUBs did not impair Met receptor, PI3K, or MAPK activation, indicating specific roles in downstream signaling.

Conclusions:

  • The ubiquitin system, regulated by DUBs, is extensively involved in multiple stages of Met receptor activation and signaling.
  • There is significant crosstalk between ubiquitination-based and phosphorylation-based signal transduction pathways in Met signaling.
  • Targeting specific DUBs with small-molecule inhibitors presents a potential therapeutic strategy to inhibit metastasis.

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