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Mechanism of IRSp53 inhibition by 14-3-3

David J Kast1,2, Roberto Dominguez3

  • 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Nature Communications
|January 31, 2019
PubMed

Insights

14-3-3 proteins inhibit IRSp53, a key regulator of cell motility and neuronal development, by binding to phosphorylation sites. This binding blocks IRSp53

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Filopodia are crucial for neuronal development and cell migration.
  • IRSp53 (Insulin receptor substrate p53) is a key regulator of filopodia dynamics.
  • IRSp53 links Rho-GTPase signaling to cytoskeleton and membrane remodeling.

Purpose of the Study:

  • To elucidate the structural and functional mechanisms of 14-3-3 protein-mediated inhibition of IRSp53.
  • To understand how phosphorylation regulates IRSp53 activity and its interactions.

Main Methods:

  • Phosphoproteomics to identify phosphorylation sites.
  • Quantitative binding assays to measure protein interactions.
  • Crystallography to determine structural basis of binding.
  • Bicistronic expression for heterodimer formation.
  • FRET-sensor assay to monitor conformational changes.
  • In vitro assays to assess membrane binding.

Main Results:

  • 14-3-3 proteins bind to two pairs of phosphorylation sites on IRSp53.
  • Each IRSp53 subunit binds one 14-3-3 dimer independently.
  • Binding of 14-3-3 induces conformational changes opposite to those induced by activators like Cdc42.
  • 14-3-3 binding inhibits IRSp53's ability to bind to membranes.

Conclusions:

  • Phosphorylation-dependent binding of 14-3-3 proteins inhibits IRSp53 function.
  • This inhibition mechanism involves blocking membrane binding and interactions with Cdc42 and downstream effectors.
  • Understanding this regulation is vital for neuronal development and cell motility research.

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