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Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse

Nature Neuroscience volume 4, pages 367–373 (2001)Cite this article

Abstract

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

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Figure 1: Increase in phosphorylation of cofilin by Sema 3A treatment.
Figure 2: Distribution and quantitation of actin filaments, phosphorylated cofilin and total cofilin at a growth cone treated with Sema 3A.
Figure 3: Inhibition of Sema 3A activity by S3-peptide.
Figure 4: Effects of dominant-negative (DN)-LIM-kinase on growth cone collapse by Sema 3A.
Figure 5: Effects of constitutively active (CA)-LIM-kinase on growth cone morphology.
Figure 6: Effects of LIM-kinase activity on outgrowth of axons from DRG explants.

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References

  1. Tessier-Lavigne, M. & Goodman, C. S. The molecular biology of axon guidance. Science 274, 1123–1133 (1996).

    Article  CAS  Google Scholar 

  2. Takahashi, T. et al. Plexin-Neuropilin-1 complexes form functional semaphorin-3A receptors. Cell 99, 59–69 (1999).

    Article  CAS  Google Scholar 

  3. Tamagnone, L. et al. Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 99, 71–80 (1999).

    Article  CAS  Google Scholar 

  4. Goshima, Y., Nakamura, F., Strittmatter, P. & Strittmatter, S. M. Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33. Nature 376, 509–514 (1995).

    Article  CAS  Google Scholar 

  5. Minturn, J. E., Fryer, H. J. L., Geschwind, D. H. & Hockfield, S. TOAD-64, a gene expressed early in neuronal differentiation in the rat, is related to unc-33, a C. elegans gene involved in axon outgrowth. J. Neurosci. 15, 6757–6766 (1995).

    Article  CAS  Google Scholar 

  6. Byk, T., Dobransky, T., Cifuentes-Diaz, C. & Sobel, A. Identification and molecular characterization of unc-33-like phosphoprotein (Ulip), a putative mammalian homologue of the axonal guidance-associated unc-33 gene product. J. Neurosci. 15, 688–701 (1995).

    Google Scholar 

  7. Jin, Z. & Strittmatter, S. M. Rac1 mediates collapsin-1-induced growth cone collapse. J. Neurosci. 17, 6256–6263 (1997).

    Article  CAS  Google Scholar 

  8. Kuhn, T. B., Brown, M. D., Wilcox, C. L., Raper, J. A. & Bamburg, J. R. Myelin and coppalsin-1 induce motor neuron growth cone collapse through different pathways: Inhibition of collapse by opposing mutants of rac1. J. Neurosci. 19, 1965–1975 (1999).

    Article  CAS  Google Scholar 

  9. Vastrik, I., Eickholt, B. J., Walsh, F. S., Ridley, A. & Doherty, P. Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17–32. Curr. Biol. 9, 991–998 (1999).

    Article  CAS  Google Scholar 

  10. Goshima, Y. et al. A novel action of collapsin: collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse. J. Neurobiol. 33, 316–328 (1997).

    Article  CAS  Google Scholar 

  11. Polleux, F., Morrow, T. & Ghosh, A. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404, 567–573 (2000).

    Article  CAS  Google Scholar 

  12. Fan, J., Mansfield, S. G., Redmond, T., Gordon-Weeks, P. R. & Raper, J. A. The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor. J. Cell Biol. 121, 867–878 (1993).

    Article  CAS  Google Scholar 

  13. Arber, S. et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393, 805–809 (1998).

    Article  CAS  Google Scholar 

  14. Yang, N. et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393, 809–812 (1998).

    Article  CAS  Google Scholar 

  15. Bamburg, J. R. Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu. Rev. Cell Dev. Biol. 15, 185–230 (1999).

    Article  CAS  Google Scholar 

  16. Carlier, M. F., Ressad, F. & Pantaloni, D. Control of actin dynamics in cell motility. Role of ADF/cofilin. J. Biol. Chem. 274, 33827–33830 (1999).

    Article  CAS  Google Scholar 

  17. Moriyama, K., Iida, K. & Yahara, I. Phosphorylation of Ser-3 of cofilin regulates its essential function on actin. Genes Cells 1, 73–86 (1996).

    Article  CAS  Google Scholar 

  18. Agnew, B. J., Minamide, L. S. & Bamburg, J. R. Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. J. Biol. Chem. 270, 17582–17587 (1995).

    Article  CAS  Google Scholar 

  19. Edwards, D. C., Sanders, L. C., Bokoch, G. M. & Gill, G. N. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signaling to actin cytoskeletal dynamics. Nat. Cell Biol. 1, 253–259 (1999).

    Article  CAS  Google Scholar 

  20. Maekawa, M. et al. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895–898 (1999).

    Article  CAS  Google Scholar 

  21. Ohashi, K. et al. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at Threonine 508 within the activation loop. J. Biol. Chem. 275, 3577–3582 (2000).

    Article  CAS  Google Scholar 

  22. Proschel, C., Blouin, M.-J., Gutowski, N. J., Ludwig, R. & Noble, M. Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro. Oncogene 11, 1271–1281 (1995).

    CAS  PubMed  Google Scholar 

  23. Tassabehji, M. et al. LIM-kinase deleted in Williams syndrome. Nat. Genet. 13, 272–273 (1996).

    Article  CAS  Google Scholar 

  24. Bamburg, J. R. & Bray, D. Distribution and cellular localization of actin depolymerizing factor. J. Cell Biol. 105, 2817–2825 (1987).

    Article  CAS  Google Scholar 

  25. Moriyama, K. & Yahara, I. Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix. EMBO J. 18, 6752–6761 (1999).

    Article  CAS  Google Scholar 

  26. Yonezawa, N., Nishida, E., Iida, K., Yahara, I. & Sakai, H. Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides. J. Biol. Chem. 265, 8382–8386 (1990).

    CAS  PubMed  Google Scholar 

  27. Aizawa, H., Sutoh, K. & Yahara, I. Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium. J. Cell Biol. 132, 335–344 (1996).

    Article  CAS  Google Scholar 

  28. Lappalainen, P. & Drubin, D. G. Cofilin promotes rapid actin filament turnover in vivo. Nature 388, 78–82 (1997).

    Article  CAS  Google Scholar 

  29. Abe, H., Obinata, T., Minamide, L. S. & Bamburg, J. R. Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development. J. Cell Biol. 132, 871–885 (1996).

    Article  CAS  Google Scholar 

  30. Prochiantz, A. Getting hydrophilic compounds into cells: lessons from homeopeptides. Curr. Opin. Neurobiol. 6, 629–634 (1996).

    Article  CAS  Google Scholar 

  31. Edwards, D. C. & Gill, G. N. Structural features of LIM kinase that control effects on the actin cytoskeleton. J. Biol. Chem. 274, 11352–11361 (1999).

    Article  CAS  Google Scholar 

  32. Machesky, L. M. Rocket-based motility: a universal mechanism? Nat. Cell Biol. 1, E29–E31 (1999).

    Article  CAS  Google Scholar 

  33. Bailly, M. et al. Relationship between Arp2/3 complex and the barbed ends of actin filaments at the leading edge of carcinoma cells after epidermal growth factor stimulation. J. Cell Biol. 145, 331–345 (1999).

    Article  CAS  Google Scholar 

  34. 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 

  35. 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 

  36. Winter, D., Lechler, T. & Li, R. Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein. Curr. Biol. 9, 501–504 (1999).

    Article  CAS  Google Scholar 

  37. Welch, M. D., Iwamatsu, A. & Mitchison, T. J. Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385, 265–269 (1997).

    Article  CAS  Google Scholar 

  38. Pantaloni, D. & Carlier, M. F. How profilin promotes actin filament assembly in the presence of thymosin beta 4. Cell 75, 1007–1014 (1993).

    Article  CAS  Google Scholar 

  39. Beckerle, M. C. Spatial control of actin filament assembly: lessons from Listeria. Cell 95, 741–748 (1998).

    Article  CAS  Google Scholar 

  40. Iida, K. et al. Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein. Gene 124, 115–120 (1993).

    Article  CAS  Google Scholar 

  41. Moon, A. L., Janmey, P. A., Louie, K. A. & Drubin, D. G. Cofilin is an essential component of the yeast cortical cytoskeleton. J. Cell Biol. 120, 421–435 (1993).

    Article  CAS  Google Scholar 

  42. Rosenblatt, J. & Mitchison, T. J. Actin, cofilin and cognition. Nature 393, 739–740 (1998).

    Article  CAS  Google Scholar 

  43. Ambach, A. et al. The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes. Eur. J. Immunol. 30, 3422–3431 (2000).

    Article  CAS  Google Scholar 

  44. Song, H. et al. Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518 (1998).

    Article  CAS  Google Scholar 

  45. Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998).

    Article  CAS  Google Scholar 

  46. Goshima, Y. et al. Growth cone neuropilin-1 mediates collapsin-1/sema III facilitation of antero- and retrograde axoplasmic transport. J. Neurobiol. 39, 579–589 (1999).

    Article  CAS  Google Scholar 

  47. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  48. Towbin, H., Staehelen, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 74, 4350–4354 (1979).

    Article  Google Scholar 

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Acknowledgements

We thank S. Ohno (Yokohama City University School of Medicine, Japan), T.C. Saido (Riken, Japan), and members of the Yahara lab and Sehara-Fujisawa lab for their comments and suggestions. We thank H. Fujisawa (Nagoya University, Japan) and T. Nagase (Kazusa DNA Research Institute, Japan) for murine neuropilin-1 and human plexin-A2 cDNAs, respectively. We also thank A. Ghosh at Johns Hopkins University Medical School for critical reading of our manuscript.

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  1. Hiroyuki Aizawa & Yoko Sekine-Aizawa

    Present address: Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland, 21205, USA

Authors and Affiliations

  1. Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, 113-8613, Tokyo, Japan

    Hiroyuki Aizawa, Shuji Wakatsuki, Ai Ishii, Kenji Moriyama, Atsuko Sehara-Fujisawa & Ichiro Yahara

  2. Department of Pharmacology, Yokohama City University School of Medicine, Yokohama, 236-0004, Japan

    Yukio Sasaki & Yoshio Goshima

  3. Biological Institute, Graduate School of Science, Tohoku University, Sendai, 980-8578, Japan

    Kazumasa Ohashi & Kensaku Mizuno

  4. Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Wako-shi, Saitama, 351-0198, Japan

    Yoko Sekine-Aizawa

  5. CREST, Japan Science and Technology Corporation (JST), Kawaguchi, 332-0012, Japan

    Yoshio Goshima

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Correspondence to Hiroyuki Aizawa.

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Aizawa, H., Wakatsuki, S., Ishii, A. et al. Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nat Neurosci 4, 367–373 (2001). https://doi.org/10.1038/86011

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