Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo
| العنوان: | Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo |
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| المؤلفون: | Shashi Prakash Singh, Peter A. Thomason, Matthias Schaks, Wulf Blankenfeldt, Robert H. Insall, Frieda Kage, Klemens Rottner, Anika Steffen, Theresia E. B. Stradal, Thomas Klünemann |
| المساهمون: | HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. |
| المصدر: | Current Biology Current biology : CB |
| بيانات النشر: | Cell Press, 2018. |
| سنة النشر: | 2018 |
| مصطلحات موضوعية: | 0301 basic medicine, rac1 GTP-Binding Protein, Protein Conformation, WAVE regulatory complex, Arp2/3 complex, RAC1, Nerve Tissue Proteins, GTPase, macromolecular substances, lamellipodium, migration, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Mice, protrusion, Cell Movement, Cell Line, Tumor, Tumor Cells, Cultured, Animals, Dictyostelium, Rho-GTPase, Pseudopodia, Binding site, CRISPR/CAS9, Adaptor Proteins, Signal Transducing, biology, Neuropeptides, filopodium, Actins, Cell biology, Wiskott-Aldrich Syndrome Protein Family, rac GTP-Binding Proteins, A-site, 030104 developmental biology, Multiprotein Complexes, biology.protein, Lamellipodium, CRISPR-Cas Systems, General Agricultural and Biological Sciences, Filopodia |
| الوصف: | Summary Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium. Graphical Abstract Highlights • WRC harbors two Rac binding sites, A and D, with distinct functions in vivo • A site is the major WRC activation site • Rac-D site interaction optimizes WAVE-mediated actin filament assembly • Both sites are obligatory for WRC activation, but not its lamellipodial accumulation Schaks et al. use gene disruption combined with re-expression of point mutants of the Sra-1/PIR121 subunit of mammalian WRC or the PIR ortholog in Dictyostelium to unravel differential functions of the two Rac interaction surfaces on WRC in vivo. These functions are crucial to both lamellipod/pseudopod protrusion and cell-migration efficiency. |
| اللغة: | English |
| تدمد: | 1879-0445 0960-9822 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::62c15093905b2a64a813c6182601b78fTest http://europepmc.org/articles/PMC6264382Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....62c15093905b2a64a813c6182601b78f |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 18790445 09609822 |
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