The dynamic polymerization of actin monomers into filament networks determines cell shape, directs intracellular organization and drives cell motility. Several cellular pathways regulating actin assembly converge on the Arp2/3 complex, a protein assembly that nucleates new actin filaments. The Arp2/3 complex is activated by N-WASP and WAVE proteins, which respond to upstream signals and instruct when and where actin networks are assembled. Unraveling the mechanisms by which N-WASP and WAVE integrate multiple signals is essential to understand how actin assembly is controlled as a coordinated process.
PI(4,5)P2 and Cdc42 synergistically activate N-WASP, promoting actin polymerization through the Arp2/3 complex. However, we found that a novel protein, transducer of Cdc42-dependent actin assembly (Toca-1), is required for actin polymerization induced by Cdc42 in Xenopus egg extracts, even though Cdc42 alone can activate isolated, recombinant N-WASP in reactions of purified proteins. We found that native N-WASP purified from Xenopus egg extracts is in a stoichiometric complex with the WASP interacting protein (WIP). WIP suppresses activation of N-WASP, and Toca-1 is required to mediate the activation of purified native N-WASP-WIP complex by Cdc42. These results establish an additional layer of regulation of N-WASP imposed by formation of the complex with WIP, and define the role of Toca-1 and other related proteins in regulating actin assembly.
WAVE proteins are found in large heteropentameric complexes whose role in regulating WAVE function is currently unclear. We found that purified native WAVE1 and WAVE2 complexes are basally inactive, and that previous reports of constitutive activity appear to be artifacts of their instability in vitro. Purified complexes cannot be activated by Rac or Nck as previously proposed. Instead, activation of the WAVE2 complex requires simultaneous binding to prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation. The WAVE2 complex can be activated fully in a highly cooperative process on the membrane surface. Activation most likely happens through allosteric changes in the complex, and not simply through its recruitment or through dissociation of its constituent subunits. These results explain how the WAVE complex integrates coincident signals to promote localized actin nucleation during morphogenesis and cell motility.
|Advisor:||Kirschner, Marc W.|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 70/07, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Actin assembly, Cytoskeleton, N-WASP, WAVE protein|
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