An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, we describe the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome.
Summary WAVE proteins link upstream signals to actin nucleation by activating the Arp2/3 complex and are at the core of regulatory pathways driving membrane protrusion. They are found in heteropentameric complexes whose role in regulating WAVE function is presently unclear. Here we demonstrate that purified native WAVE complexes are basally inactive; previous reports of constitutive activity are artifacts of in vitro manipulation. Further, the native complexes are not activated by Rac alone. Activation of the WAVE2 complex requires simultaneous interactions with prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation. Together these signals promote full activation in a highly cooperative process on the membrane surface, by inducing an allosteric change in the complex rather than by simple recruitment or by dissociation of the subunits. These results explain how the WAVE complex can integrate coincident signals to promote localized actin nucleation during cell motility.
The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.DOI: http://dx.doi.org/10.7554/eLife.21459.001
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