Allosteric N-WASP activation by an inter-SH3 domain linker in Nck

Proc Natl Acad Sci U S A. 2015 Nov 24;112(47):E6436-45. doi: 10.1073/pnas.1510876112. Epub 2015 Nov 9.


Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott-Aldrich-syndrome protein (N-WASP) and the Arp2/3 complex. An inactive conformation of N-WASP is stabilized by intramolecular contacts between the GTPase binding domain (GBD) and the C helix of the verprolin-homology, connector-helix, acidic motif (VCA) segment. Multiple SH3 domain-containing adapter proteins can bind and possibly activate N-WASP, but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2/3 complex. Here, we have used purified components to reconstitute a signaling cascade driven by membrane-localized Src homology 3 (SH3) adapters and N-WASP, resulting in the assembly of dynamic actin networks. Among six SH3 adapters tested, Nck was the most potent activator of N-WASP-driven actin assembly. We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains. This linker sequence, reminiscent of bacterial virulence factors, directly engages the N-WASP GBD and competes with VCA binding. Our results suggest that animals, like pathogenic bacteria, have evolved peptide motifs that allosterically activate N-WASP, leading to localized actin nucleation on cellular membranes.

Keywords: N-WASP; Nck; SH3 adapter; actin cytoskeleton; signal transduction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Adaptor Proteins, Signal Transducing / chemistry*
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Allosteric Regulation
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / metabolism
  • Mice
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Oncogene Proteins / chemistry*
  • Oncogene Proteins / metabolism*
  • Protein Binding
  • Protein Structure, Secondary
  • Rats
  • Structure-Activity Relationship
  • Wiskott-Aldrich Syndrome Protein, Neuronal / metabolism*
  • src Homology Domains*


  • Actins
  • Adaptor Proteins, Signal Transducing
  • Membrane Proteins
  • Mutant Proteins
  • Nck protein
  • Oncogene Proteins
  • Wiskott-Aldrich Syndrome Protein, Neuronal
  • nephrin