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Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors

David J Kast1, Changsong Yang2, Andrea Disanza3

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

Nature Structural & Molecular Biology
|March 4, 2014
PubMed

Insights

The Rho GTPase effector IRSp53 regulates filopodia formation. Its activation requires synergistic binding of Cdc42 and effector proteins, revealing a novel regulatory mechanism.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • IRSp53 is a Rho family GTPase effector crucial for filopodia formation and neuronal development.
  • Its precise regulatory mechanism remains largely unelucidated.
  • IRSp53 possesses a BAR domain, an unconventional CRIB motif (CRIB-PR), and an SH3 domain.

Purpose of the Study:

  • To elucidate the regulatory mechanism of IRSp53.
  • To investigate the synergistic activation of IRSp53 by Cdc42 and effector proteins.
  • To determine the structural basis of IRSp53-Cdc42 interaction.

Main Methods:

  • Fluorescence reporter assay to study IRSp53 conformation.
  • Co-expression of IRSp53, Cdc42, and Eps8 in human cells.
  • X-ray crystallography to determine the structure of Cdc42 bound to CRIB-PR.
  • Site-directed mutagenesis to assess the functional impact of mutations.

Main Results:

  • Human IRSp53 exists in a closed, inactive conformation.
  • IRSp53 activation occurs synergistically upon binding of human Cdc42 to CRIB-PR and effector proteins (e.g., Eps8) to the SH3 domain.
  • Crystal structure reveals a novel binding mode between Cdc42 and the CRIB-PR.
  • Mutations disrupting autoinhibition and Cdc42 binding impair filopodia formation in cells.

Conclusions:

  • IRSp53 activation follows a combinatorial mechanism.
  • Synergistic binding of Cdc42 and effectors is essential for IRSp53 function.
  • The findings provide new insights into Rho GTPase effector regulation and filopodia dynamics.

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