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A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex

Nature Structural & Molecular Biology volume 10, pages 591–598 (2003)Cite this article

Abstract

Members of the Wiskott-Aldrich syndrome protein (WASP) family link Rho GTPase signaling pathways to the cytoskeleton through a multiprotein assembly called Arp2/3 complex. The C-terminal VCA regions (verprolin-homology, central hydrophobic, and acidic regions) of WASP and its relatives stimulate Arp2/3 complex to nucleate actin filament branches. Here we show by differential line broadening in NMR spectra that the C (central) and A (acidic) segments of VCA domains from WASP, N-WASP and Scar bind Arp2/3 complex. The C regions of these proteins have a conserved sequence motif consisting of hydrophobic residues and an arginine residue. Point mutations in this conserved sequence motif suggest that it forms an amphipathic helix that is required in biochemcial assays for activation of Arp2/3 complex. Key residues in this motif are buried through contacts with the GTPase binding domain in the autoinhibited structure of WASP and N-WASP, indicating that sequestration of these residues is an important aspect of autoinhibition.

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Figure 1: Schematic diagram showing domain organization in the WASP family proteins.
Figure 2: NMR line-broadening experiments.
Figure 3: 1H-15N HSQC peak intensities of VCA peptides in the presence and absence of Arp2/3 complex.
Figure 4: Conserved motif in the C region.
Figure 5: Actin-filament nucleation by Arp2/3 complex stimulated by VCA peptides.
Figure 6: Residues necessary for activation of Arp2/3 complex are buried in the autoinhibited state of WASP/N-WASP.

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References

  1. Borisy, G.G. & Svitkina, T.M. Actin machinery: pushing the envelope. Curr. Opin. Cell Biol. 12, 104–112 (2000).

    Article  CAS  Google Scholar 

  2. Pantaloni, D., Le Clainche, C. & Carlier, M.F. Mechanism of actin-based motility. Science 292, 1502–1506 (2001).

    Article  CAS  Google Scholar 

  3. Svitkina, T.M. & Borisy, G.G. Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J. Cell Biol. 145, 1009–1026 (1999).

    Article  CAS  Google Scholar 

  4. Higgs, H.N. & Pollard, T.D. Regulation of actin filament network formation through Arp2/3 complex: activation by a diverse array of proteins. Annu. Rev. Biochem. 70, 649–676 (2001).

    Article  CAS  Google Scholar 

  5. Machesky, L.M. & Gould, K.L. The Arp2/3 complex: a multifunctional actin organizer. Curr. Opin. Cell Biol. 11, 117–121 (1999).

    Article  CAS  Google Scholar 

  6. Caron, E. Regulation of Wiskott-Aldrich syndrome protein and related molecules. Curr. Opin. Cell Biol. 14, 82–87 (2002).

    Article  CAS  Google Scholar 

  7. Weaver, A.M., Young, M.E., Lee, W.L. & Cooper, J.A. Integration of signals to the Arp2/3 complex. Curr. Opin. Cell Biol. 15, 23–30 (2003).

    Article  CAS  Google Scholar 

  8. Frischknecht, F. & Way, M. Surfing pathogens and the lessons learned for actin polymerization. Trends Cell Biol. 11, 30–38 (2001).

    Article  CAS  Google Scholar 

  9. Ochs, H.D. The Wiskott-Aldrich syndrome. Clin. Rev. Allergy Immunol. 20, 61–86 (2001).

    Article  CAS  Google Scholar 

  10. Devriendt, K. et al. Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat. Genet. 27, 313–317 (2001).

    Article  CAS  Google Scholar 

  11. Higgs, H.N., Blanchoin, L. & Pollard, T.D. Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. Biochemistry 38, 15212–15222 (1999).

    Article  CAS  Google Scholar 

  12. Miki, H., Miura, K. & Takenawa, T. N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J. 15, 5326–5335 (1996).

    Article  Google Scholar 

  13. Marchand, J.B., Kaiser, D.A., Pollard, T.D. & Higgs, H.N. Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex. Nat. Cell Biol. 3, 76–82 (2001).

    Article  CAS  Google Scholar 

  14. Miki, H. & Takenawa, T. Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization. Biochem. Biophys. Res. Comm. 243, 73–78 (1998).

    Article  CAS  Google Scholar 

  15. Yamaguchi, H. et al. Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP. Proc. Natl. Acad. Sci. USA 97, 12631–12636 (2000).

    Article  CAS  Google Scholar 

  16. Rohatgi, R. et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221–231 (1999).

    Article  CAS  Google Scholar 

  17. Sasaki, N., Miki, H. & Takenawa, T. Arp2/3 complex-independent actin regulatory function of WAVE. Biochem. Biophys. Res. Commun. 272, 386–390 (2000).

    Article  CAS  Google Scholar 

  18. Machesky, L.M. & Insall, R.H. Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr. Biol. 8, 1347–1356 (1998).

    Article  CAS  Google Scholar 

  19. Eden, S. & Kirschner, M.W. Inhibition of Scar/WAVE proteins by the Pir121/NAP-1 Dimer is relieved by Rac. Nature 418, 790–793 (2002).

    Article  CAS  Google Scholar 

  20. Kim, A.S., Kakalis, L.T., Abdul-Manan, N., Liu, G.A. & Rosen, M.K. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 404, 151–158 (2000).

    Article  CAS  Google Scholar 

  21. Rohatgi, R., Ho, H.Y. & Kirschner, M.W. Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate. J. Cell Biol. 150, 1299–1310 (2000).

    Article  CAS  Google Scholar 

  22. Higgs, H.N. & Pollard, T.D. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J. Cell Biol. 150, 1311–1320 (2000).

    Article  CAS  Google Scholar 

  23. Buck, M., Xu, W. & Rosen, M.K. Global disruption of the WASP autoinhibited fold on Cdc42 binding. Ligand displacement as a novel method to monitor amide hydrogen exchange. Biochemistry 40, 14115–14122 (2001).

    Article  CAS  Google Scholar 

  24. Prehoda, K.E., Scott, J.A., Mullins, R.D. & Lim, W.A. Integration of multiple signals through cooperative regulation of the N- WASP-Arp2/3 complex. Science 290, 801–806 (2000).

    Article  CAS  Google Scholar 

  25. Muhandiram, D.R. & Kay, L.E. Gradient-enhanced triple-resonance three-dimensional NMR experiments with improved sensitivity. J. Magn. Reson. B 103, 203–216 (1994).

    Article  CAS  Google Scholar 

  26. Panchal, S.C., Bhavesh, N.S. & Hosur, R.V. Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins. J. Biomol. NMR 20, 135–147 (2001).

    Article  CAS  Google Scholar 

  27. Wishart, D.S. & Sykes, B.D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4, 171–180 (1994).

    Article  CAS  Google Scholar 

  28. Zalevsky, J., Grigorova, I. & Mullins, R.D. Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex. J. Biol. Chem. 276, 3468–3475 (2001).

    Article  CAS  Google Scholar 

  29. Matsuo, H. et al. Identification by NMR spectroscopy of residues at contact surfaces in large, slowly exchanging macromolecular complexes. J. Am. Chem. Soc. 121, 9903–9904 (1999).

    Article  CAS  Google Scholar 

  30. Pistor, S. et al. Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex. J. Cell Sci. 113, 3277–3287. (2000).

    CAS  PubMed  Google Scholar 

  31. Machesky, L.M. et al. Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. Proc. Natl. Acad. Sci. USA 96, 3739–3744 (1999).

    Article  CAS  Google Scholar 

  32. Pollard, T.D. & Cooper, J.A. Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation. Biochemistry 23, 6631–6641 (1984).

    Article  CAS  Google Scholar 

  33. Notarangelo, L.D. et al. Missense mutations of the WASP gene cause intermittent X-linked thrombocytopenia. Blood 99, 2268–2269. (2002).

    Article  CAS  Google Scholar 

  34. Prehoda, K.E., Lee, D.J. & Lim, W.A. Structure of the enabled/VASP homology 1 domain-peptide complex: a key component in the spatial control of actin assembly. Cell 97, 471–480 (1999).

    Article  CAS  Google Scholar 

  35. Zalevsky, J., Lempert, L., Kranitz, H. & Mullins, R.D. Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities. Curr. Biol. 11, 1903–1913 (2001).

    Article  CAS  Google Scholar 

  36. Robinson, R.C. et al. Crystal structure of arp2/3 complex. Science 294, 1679–1684 (2001).

    Article  CAS  Google Scholar 

  37. Volkmann, N. et al. Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science 293, 2456–2459 (2001).

    Article  CAS  Google Scholar 

  38. Le Clainche, C., Didry, D., Carlier, M.F. & Pantaloni, D. Activation of Arp2/3 complex by Wiskott-Aldrich syndrome protein is linked to enhanced binding of ATP to Arp2. J. Biol. Chem. 276, 46689–46692 (2001).

    Article  CAS  Google Scholar 

  39. Dayel, M.J., Holleran, E.A. & Mullins, R.D. Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments. Proc. Natl. Acad. Sci. USA 98, 14871–14876 (2001).

    Article  CAS  Google Scholar 

  40. MacLean-Fletcher, S. & Pollard, T.D. Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biochem. Biophys. Res. Comm. 96, 18–27 (1980).

    Article  CAS  Google Scholar 

  41. Yamazaki, T., Lee, W., Arrowsmith, C., Muhandiriam, D. & Kay, L. A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J. Am. Chem. Soc. 116, 11655–11666 (1994).

    Article  CAS  Google Scholar 

  42. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

  43. Johnson, B.A. & Blevins, R.A. NMRView: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Padrick for writing Mathematica scripts used in the analysis of anisotropy data. This work was supported by grants from the US National Institutes of Health (NIH) and Welch Foundation to M.K.R. and an NIH research grant to T.D.P.

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Authors and Affiliations

  1. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Sanjay C Panchal, Eduardo Torres & Michael K Rosen

  2. The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, 92037, California, USA

    Donald A Kaiser

  3. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, 10021, New York, USA

    Eduardo Torres

  4. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, 06520, Connecticut, USA

    Thomas D Pollard

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Correspondence to Michael K Rosen.

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Panchal, S., Kaiser, D., Torres, E. et al. A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex. Nat Struct Mol Biol 10, 591–598 (2003). https://doi.org/10.1038/nsb952

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