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Cited by 85Protein Kinase Regulation and GTPase SignalingCellular Mechanics and InteractionsCellular transport and secretion

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo

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Schaks, Matthias, Singh, Shashi P., Kage, Frieda, Thomason, Peter, Klünemann, Thomas, Steffen, Anika, Blankenfeldt, Wulf, Stradal, Theresia E. +2 more·2018·Current Biology
DOI 10.1016/j.cub.2018.10.002PMC PMC6264382
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Abstract

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.

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  • Rac1 binds to two sites on CYFIP in the WAVE complex, leading to WAVE activation.

    “Rac1 binds to two sites on CYFIP in the WAVE complex, leading to WAVE activation28,30–32.”

    From The WAVE complex in developmental and adulthood brain disorders (2025) · cites this paper as [32]

    The Results and Discussion section identifies two Rac-binding sites on Sra-1/CYFIP and says Rac binding releases the WAVE WCA domain, enabling activation. It also finds that the A site is crucial for allosteric activation, whereas the D site is dispensable for activation. The claim is therefore supported on binding and activation generally, but not if it implies that binding at both sites drives activation. (LLM, conf=0.87)

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  1. [1]Molinie N. Gautreau A. The Arp2/3 regulatory system and its deregulation in cancer Physiol. Rev. 98 2018 215 238 29212790
  2. [2]Rottner K. Faix J. Bogdan S. Linder S. Kerkhoff E. Actin assembly mechanisms at a glance J. Cell Sci. 130 2017 3427 3435 29032357
  3. [3]Chen B. Chou H.T. Brautigam C.A. Xing W. Yang S. Henry L. Doolittle L.K. Walz T. Rosen M.K. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites eLife 6 2017 e29795 28949297 PMC5614565
  4. [4]Chen Z. Borek D. Padrick S.B. Gomez T.S. Metlagel Z. Ismail A.M. Umetani J. Billadeau D.D. Otwinowski Z. Rosen M.K. Structure and control of the actin regulatory WAVE complex Nature 468 2010 533 538 21107423 PMC3085272
  5. [5]Alekhina O. Burstein E. Billadeau D.D. Cellular functions of WASP family proteins at a glance J. Cell Sci. 130 2017 2235 2241 28646090 PMC5536917
  6. [6]Derivery E. Fink J. Martin D. Houdusse A. Piel M. Stradal T.E. Louvard D. Gautreau A. Free Brick1 is a trimeric precursor in the assembly of a functional Wave complex PLoS ONE 3 2008 e2462 18560548 PMC2413427
  7. [7]Innocenti M. Zucconi A. Disanza A. Frittoli E. Areces L.B. Steffen A. Stradal T.E. Di Fiore P.P. Carlier M.F. Scita G. Abi1 is essential for the formation and activation of a WAVE2 signalling complex Nat. Cell Biol. 6 2004 319 327 15048123
  8. [9]Rogers S.L. Wiedemann U. Stuurman N. Vale R.D. Molecular requirements for actin-based lamella formation in Drosophila S2 cells J. Cell Biol. 162 2003 1079 1088 12975351 PMC2172842
  9. [10]Steffen A. Rottner K. Ehinger J. Innocenti M. Scita G. Wehland J. Stradal T.E. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation EMBO J. 23 2004 749 759 14765121 PMC380996
  10. [11]Bear J.E. Rawls J.F. Saxe C.L. III SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development J. Cell Biol. 142 1998 1325 1335 9732292 PMC2149354
  11. [12]Blagg S.L. Stewart M. Sambles C. Insall R.H. PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium Curr. Biol. 13 2003 1480 1487 12956949
  12. [13]Ibarra N. Blagg S.L. Vazquez F. Insall R.H. Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways Curr. Biol. 16 2006 717 722 16581519
  13. [14]King J.S. Veltman D.M. Georgiou M. Baum B. Insall R.H. SCAR/WAVE is activated at mitosis and drives myosin-independent cytokinesis J. Cell Sci. 123 2010 2246 2255 20530573 PMC2886745
  14. [15]Litschko C. Linkner J. Brühmann S. Stradal T.E.B. Reinl T. Jänsch L. Rottner K. Faix J. Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes Eur. J. Cell Biol. 96 2017 715 727 28889942
  15. [16]Dubielecka P.M. Ladwein K.I. Xiong X. Migeotte I. Chorzalska A. Anderson K.V. Sawicki J.A. Rottner K. Stradal T.E. Kotula L. Essential role for Abi1 in embryonic survival and WAVE2 complex integrity Proc. Natl. Acad. Sci. USA 108 2011 7022 7027 21482783 PMC3084068
  16. [17]Leithner A. Eichner A. Müller J. Reversat A. Brown M. Schwarz J. Merrin J. de Gorter D.J. Schur F. Bayerl J. Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes Nat. Cell Biol. 18 2016 1253 1259 27775702
  17. [18]Soderling S.H. Langeberg L.K. Soderling J.A. Davee S.M. Simerly R. Raber J. Scott J.D. Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice Proc. Natl. Acad. Sci. USA 100 2003 1723 1728 12578964 PMC149900
  18. [19]Yamazaki D. Suetsugu S. Miki H. Kataoka Y. Nishikawa S. Fujiwara T. Yoshida N. Takenawa T. WAVE2 is required for directed cell migration and cardiovascular development Nature 424 2003 452 456 12879075
  19. [20]Suetsugu S. Yamazaki D. Kurisu S. Takenawa T. Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration Dev. Cell 5 2003 595 609 14536061
  20. [21]Hahne P. Sechi A. Benesch S. Small J.V. Scar/WAVE is localised at the tips of protruding lamellipodia in living cells FEBS Lett. 492 2001 215 220 11257497
  21. [22]Pollitt A.Y. Insall R.H. Abi mutants in Dictyostelium reveal specific roles for the SCAR/WAVE complex in cytokinesis Curr. Biol. 18 2008 203 210 18261908
  22. [23]Lebensohn A.M. Kirschner M.W. Activation of the WAVE complex by coincident signals controls actin assembly Mol. Cell 36 2009 512 524 19917258 PMC2818508
  23. [24]Oikawa T. Yamaguchi H. Itoh T. Kato M. Ijuin T. Yamazaki D. Suetsugu S. Takenawa T. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia Nat. Cell Biol. 6 2004 420 426 15107862
  24. [25]Steffen A. Ladwein M. Dimchev G.A. Hein A. Schwenkmezger L. Arens S. Ladwein K.I. Margit Holleboom J. Schur F. Victor Small J. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation J. Cell Sci. 126 2013 4572 4588 23902686 PMC3817791
  25. [26]Fort L. Batista J.M. Thomason P.A. Spence H.J. Whitelaw J.A. Tweedy L. Greaves J. Martin K.J. Anderson K.I. Brown P. Fam49/CYRI interacts with Rac1 and locally suppresses protrusions Nat Cell Biol. 20 2018 1159 1171 30250061 PMC6863750
  26. [27]Chen B. Brinkmann K. Chen Z. Pak C.W. Liao Y. Shi S. Henry L. Grishin N.V. Bogdan S. Rosen M.K. The WAVE regulatory complex links diverse receptors to the actin cytoskeleton Cell 156 2014 195 207 24439376 PMC4059610
  27. [28]Squarr A.J. Brinkmann K. Chen B. Steinbacher T. Ebnet K. Rosen M.K. Bogdan S. Fat2 acts through the WAVE regulatory complex to drive collective cell migration during tissue rotation J. Cell Biol. 212 2016 591 603 26903538 PMC4772498
  28. [29]Fritz-Laylin L.K. Riel-Mehan M. Chen B.C. Lord S.J. Goddard T.D. Ferrin T.E. Nicholson-Dykstra S.M. Higgs H. Johnson G.T. Betzig E. Mullins R.D. Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes eLife 6 2017 e26990 28948912 PMC5614560
  29. [30]Veltman D.M. King J.S. Machesky L.M. Insall R.H. SCAR knockouts in Dictyostelium: WASP assumes SCAR’s position and upstream regulators in pseudopods J. Cell Biol. 198 2012 501 508 22891261 PMC3514037
  30. [31]Bieling P. Hansen S.D. Akin O. Li T.D. Hayden C.C. Fletcher D.A. Mullins R.D. WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation EMBO J. 37 2018 102 121 29141912 PMC5753033
  31. [32]Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenström P. Bridges T. Chant J. Hall A. Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade Cell 87 1996 519 529 8898204
  32. [33]Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways Mol. Cell. Biol. 17 1997 1324 1335 9032259 PMC231857
  33. [34]Pellegrin S. Mellor H. Actin stress fibres J. Cell Sci. 120 2007 3491 3499 17928305
  34. [35]Lai F.P. Szczodrak M. Block J. Faix J. Breitsprecher D. Mannherz H.G. Stradal T.E. Dunn G.A. Small J.V. Rottner K. Arp2/3 complex interactions and actin network turnover in lamellipodia EMBO J. 27 2008 982 992 18309290 PMC2265112
  35. [36]Weiner O.D. Marganski W.A. Wu L.F. Altschuler S.J. Kirschner M.W. An actin-based Wave generator organizes cell motility PLoS Biol. 5 2007 e221 17696648 PMC1945041
  36. [37]Pollitt A.Y. Blagg S.L. Ibarra N. Insall R.H. Cell motility and SCAR localisation in axenically growing Dictyostelium cells Eur. J. Cell Biol. 85 2006 1091 1098 16822579
  37. [38]Olazabal I.M. Caron E. May R.C. Schilling K. Knecht D.A. Machesky L.M. Rho-kinase and myosin-II control phagocytic cup formation during CR, but not FcgammaR, phagocytosis Curr. Biol. 12 2002 1413 1418 12194823
  38. [39]Chisholm R.L. Gaudet P. Just E.M. Pilcher K.E. Fey P. Merchant S.N. Kibbe W.A. dictyBase, the model organism database for Dictyostelium discoideum Nucleic Acids Res. 34 2006 D423 D427 16381903 PMC1347453
  39. [40]Nobes C.D. Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia Cell 81 1995 53 62 7536630
  40. [41]Kage F. Winterhoff M. Dimchev V. Mueller J. Thalheim T. Freise A. Brühmann S. Kollasser J. Block J. Dimchev G. FMNL formins boost lamellipodial force generation Nat. Commun. 8 2017 14832 28327544 PMC5364437
  41. [42]Davidson A.J. Amato C. Thomason P.A. Insall R.H. WASP family proteins and formins compete in pseudopod- and bleb-based migration J. Cell Biol. 217 2018 701 714 29191847 PMC5800805
  42. [43]Kuspa A. Loomis W.F. Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA Proc. Natl. Acad. Sci. USA 89 1992 8803 8807 1326764 PMC50009
  43. [44]Laevsky G. Knecht D.A. Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments J. Cell Sci. 116 2003 3761 3770 12890752
  44. [45]Koestler S.A. Steffen A. Nemethova M. Winterhoff M. Luo N. Holleboom J.M. Krupp J. Jacob S. Vinzenz M. Schur F. Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin Mol. Biol. Cell 24 2013 2861 2875 23885122 PMC3771948
  45. [46]de Vries S.J. van Dijk A.D. Krzeminski M. van Dijk M. Thureau A. Hsu V. Wassenaar T. Bonvin A.M. HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets Proteins 69 2007 726 733 17803234
  46. [48]van Zundert G.C.P. Melquiond A.S.J. Bonvin A.M.J.J. Integrative modeling of biomolecular complexes: HADDOCKing with cryo-electron microscopy data Structure 23 2015 949 960 25914056
  47. [49]Krauthammer M. Kong Y. Ha B.H. Evans P. Bacchiocchi A. McCusker J.P. Cheng E. Davis M.J. Goh G. Choi M. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma Nat. Genet. 44 2012 1006 1014 22842228 PMC3432702
  48. [50]Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera—a visualization system for exploratory research and analysis J. Comput. Chem. 25 2004 1605 1612 15264254

Cited by

Sorted by citations

Authors
Schaks, M., Singh, S. P., Kage, F., Thomason, P., Klünemann, T., Steffen, A., Blankenfeldt, W., Stradal, T. E., Insall, R. H., & Rottner, K.
Affiliations
  • Helmholtz Centre for Infection Research (DE)
  • Cancer Research UK Scotland Institute (GB)
Corresponding author
  • Insall, Robert H.
  • Rottner, Klemens
ORCID
Venue
Current Biology
Publisher
Elsevier BV
Published
2018-11
Volume
28
Issue
22
Pages
3674-3684.e6
ISSN
0960-9822 (ISSN-L)
DOI
10.1016/j.cub.2018.10.002
PMC
PMC6264382
OpenAlex
W2898886924
Cited by
85
References
2
Citations by year
2026: 2 · 2025: 11 · 2024: 7 · 2023: 8 · 2022: 15 · 2021: 14 · 2020: 21 · 2019: 5 · 2018: 2
FWCI
3.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Current Biology (publishedVersion, cc-by-nc-nd)
  • SSRN Electronic Journal (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • ENLIGHTEN (Jurnal Bimbingan dan Konseling Islam) (acceptedVersion, cc-by-sa)
  • HZI (Helmholtz Centre for Infection Research) (submittedVersion, cc-by-nc-sa)
Funders
  • Deutsche Forschungsgemeinschaft (GRK2223/1, RO2414/3-2)
  • Cancer Research UK (CRUK-A20017)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
Rac GTPase, WAVE regulatory complex, allosteric activation, lamellipodia, Arp2/3 complex, branched actin filaments, cell migration, Dictyostelium, Rac1, membrane recruitment
MeSH terms
RAC2 GTP-Binding Protein, Actins, Animals, Cell Movement, Dictyostelium, Nerve Tissue Proteins, Neuropeptides, Protein Conformation, Pseudopodia, Tumor Cells, Cultured, rac GTP-Binding Proteins, rac1 GTP-Binding Protein, Cell Line, Tumor, Multiprotein Complexes, Adaptor Proteins, Signal Transducing, Wiskott-Aldrich Syndrome Protein Family, Mice, CRISPR-Cas Systems

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