Regulation of WASP in Actin Signaling: From Angstroms to Microns




Cheng, Hui-Chun

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Wiskott-Aldrich syndrome protein (WASP) regulates membrane-attached force generation by promoting actin polymerization, which is key to cell motility and immune responses. Mutations in WASP lead to Wiskott-Aldrich syndrome and neutropenia. My graduate research was focused on allosteric regulation of WASP by an Enterohaemorrhagic E. coli (EHEC) effector EspFU. This study leads to novel mechanisms of WASP regulation and also sheds light on how cells conduct μm-scale assembly by orchestrating nm-scale proteins spatially and temporally. EHEC hijacks the host cytoskeleton during infection to form actin-rich ‘pedestals’ beneath the invading bacteria. The multiple repeat-containing effector EspFU stimulates pedestal formation by activating WASP, which is itself autoinhibited through interactions between a regulatory GTPase binding domain (GBD) and an activity bearing VCA region. First, I investigated how a single repeat fragment (1R) from EspFU relieves WASP autoinhibition. By determining the solution structure of a complex between the WASP GBD and 1R, I showed that EspFU binds to WASP by mimicking the C region of the VCA domain. 1R has a higher affinity toward the GBD than does VCA, allowing it to displace the VCA and activate WASP. Next, I examined the role of multiple repeats of EspFU. Surprisingly, a two-repeat fragment is much more potent in stimulating WASP-dependent actin polymerization than a single repeat at the same total repeat concentration. My colleagues and I showed that the inter-repeat cooperativity of EspFU originates from its ability to engage two active WASP molecules. Such dimers have much higher affinity (~100-fold) than monomers for the actin nucleation factor, the Arp2/3 complex. The combined mechanism enables EHEC to dominate the eukaryotic cytoskeletal machinery. These studies culminated in discovery of the hierarchical regulation of WASP proteins: allosteric activators release autoinhibition, and dimerization/oligomerization of active WASP molecules further enhances activity in promoting actin assembly by the Arp2/3 complex. Multivalency is widespread from extracellular sugar binding proteins to transmembrane receptors to cytoplasmic adapters to nuclear chromatin. Multivalent interactions play a fundamental but incompletely understood role in biology. To assess its roles in signal transduction, my colleagues and I utilized a battery of biochemical and biophysical tools to study a model system, where each multivalent molecule harbors identical modules, and a natural system, where each multivalent molecule consists of homologous modules. In the model system, I observed μm-scale droplet-like protein assembly (phase separation) upon mixing multivalent proteins above a critical concentration. The critical concentration correlates with the valency and individual module binding affinity. In the natural system, the multivalent signaling network consisting of WASP, Nck and Nephrin is necessary to maintain the structural and functional integrity of glomeruli in the kidney. Upon phosphorylation, three phosphotyrosine motifs in the cytoplasmic domain of a transmembrane scaffolding protein Nephrin recruit the C terminal SH2 domain of Nck, whose three SH3 domains interact with multiple proline-rich motifs of WASP. We also observed phase transitions in this system and found that the phase boundary position is highly dependent on the degree of nephrin phosphorylation, suggesting that kinases could induce phase transitions to remodel cellular structure. Furthermore, the phase transition here correlates with a sharp transition in the ability of WASP to stimulate actin assembly by the Arp2/3 complex. Together, this work suggests that cells may exploit multivalency as one of means of regulating the spatial organization and biochemical activity of signaling molecules in response to the environment.

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