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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

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Protein interaction screening identifies SH3RF1 as a new regulator of FAT1 protein levels

Charles E de Bock1,2, Michael R Hughes3, Kimberly Snyder3

  • 1VIB Center for the Biology of Disease, Leuven, Belgium.

FEBS Letters
|January 28, 2017
PubMed

Insights

SH3RF1 E3 ubiquitin-protein ligase negatively regulates FAT1 cadherin levels. Ablating SH3RF1 increases FAT1 protein and cell surface expression, while SH3RF1 overexpression decreases FAT1 levels, clarifying FAT1 regulation.

Area of Science:

  • Cell biology
  • Molecular biology
  • Biochemistry

Background:

  • Mutations and aberrant expression of FAT1 cadherin are linked to developmental disorders and cancers.
  • The precise regulatory mechanisms governing FAT1 and its signaling pathways are not fully elucidated.

Purpose of the Study:

  • To identify novel protein interactors of the FAT1 cytoplasmic tail to better understand its regulation and function.
  • To investigate the role of identified interactors in modulating FAT1 protein levels and cellular localization.

Main Methods:

  • Yeast two-hybrid library screening against the FAT1 cytoplasmic tail juxtamembrane region.
  • RNA interference (siRNA) to ablate SH3RF1 expression.
  • Western blotting to assess protein levels and cell surface expression.

Main Results:

  • The E3 ubiquitin-protein ligase SH3RF1 was identified as a frequent binding partner of the FAT1 cytoplasmic tail.
  • siRNA-mediated knockdown of SH3RF1 resulted in increased cellular FAT1 protein levels.
  • SH3RF1 ablation led to stabilized FAT1 expression at the cell surface.
  • Overexpression of SH3RF1 reduced overall FAT1 protein levels.

Conclusions:

  • SH3RF1 functions as a negative post-translational regulator of FAT1 protein levels.
  • SH3RF1 promotes the degradation or reduces the stability of FAT1.
  • These findings provide new insights into the molecular mechanisms controlling FAT1 cadherin homeostasis.
Keywords:
E3 ubiquitin-ligaseFAT1 cadherinSH3RF1protein half-lifeprotein-protein interactionyeast two-hybrid

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