title: Allosteric N-WASP activation by an inter-SH3 domain linker in Nck.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; The Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, MA 02543;
authors.forename: Julia
authors.initials: J
authors.lastname: Okrut
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158;
authors.forename: Sumit
authors.initials: S
authors.lastname: Prakash
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158.
authors.forename: Mark J S
authors.initials: MJ
authors.lastname: Kelly
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; The Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, MA 02543; jack.taunton@ucsf.edu.
authors.forename: Jack
authors.initials: J
authors.lastname: Taunton
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D015513:Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D013329:Structure-Activity Relationship; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; D050505:Mutant Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C113267:nephrin
keywords: N-WASP; Nck; SH3 adapter; actin cytoskeleton; signal transduction