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. 1994 Aug 1;126(3):801–810. doi: 10.1083/jcb.126.3.801

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho

PMCID: PMC2120149  PMID: 8045941

Abstract

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

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Selected References

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  1. Adamson P., Paterson H. F., Hall A. Intracellular localization of the P21rho proteins. J Cell Biol. 1992 Nov;119(3):617–627. doi: 10.1083/jcb.119.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adler R. Regulation of neurite growth in purified retina neuronal cultures: effects of PNPF, a substratum-bound, neurite-promoting factor. J Neurosci Res. 1982;8(2-3):165–177. doi: 10.1002/jnr.490080207. [DOI] [PubMed] [Google Scholar]
  3. Aktories K., Weller U., Chhatwal G. S. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 1987 Feb 9;212(1):109–113. doi: 10.1016/0014-5793(87)81566-1. [DOI] [PubMed] [Google Scholar]
  4. Amano T., Richelson E., Nirenberg M. Neurotransmitter synthesis by neuroblastoma clones (neuroblast differentiation-cell culture-choline acetyltransferase-acetylcholinesterase-tyrosine hydroxylase-axons-dendrites). Proc Natl Acad Sci U S A. 1972 Jan;69(1):258–263. doi: 10.1073/pnas.69.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baorto D. M., Mellado W., Shelanski M. L. Astrocyte process growth induction by actin breakdown. J Cell Biol. 1992 Apr;117(2):357–367. doi: 10.1083/jcb.117.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bar-Sagi D., Feramisco J. R. Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell. 1985 Oct;42(3):841–848. doi: 10.1016/0092-8674(85)90280-6. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  8. Burridge K. Are stress fibres contractile? Nature. 1981 Dec 24;294(5843):691–692. doi: 10.1038/294691a0. [DOI] [PubMed] [Google Scholar]
  9. Chardin P., Boquet P., Madaule P., Popoff M. R., Rubin E. J., Gill D. M. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davis G. E., Skaper S. D., Manthorpe M., Moonen G., Varon S. Fetal calf serum-mediated inhibition of neurite growth from ciliary ganglion neurons in vitro. J Neurosci Res. 1984;12(1):29–39. doi: 10.1002/jnr.490120104. [DOI] [PubMed] [Google Scholar]
  11. Dennerll T. J., Joshi H. C., Steel V. L., Buxbaum R. E., Heidemann S. R. Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements. J Cell Biol. 1988 Aug;107(2):665–674. doi: 10.1083/jcb.107.2.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Didsbury J., Weber R. F., Bokoch G. M., Evans T., Snyderman R. rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem. 1989 Oct 5;264(28):16378–16382. [PubMed] [Google Scholar]
  13. Dyer D., Tigyi G., Miledi R. The effect of active serum albumin on PC12 cells: I. Neurite retraction and activation of the phosphoinositide second messenger system. Brain Res Mol Brain Res. 1992 Aug;14(4):293–301. doi: 10.1016/0169-328x(92)90096-t. [DOI] [PubMed] [Google Scholar]
  14. Edwards J. G., Campbell G., Carr M., Edwards C. C. Shapes of cells spreading on fibronectin: measurement of the stellation of BHK21 cells induced by raising cyclic AMP, and of its reversal by serum and lysophosphatidic acid. J Cell Sci. 1993 Feb;104(Pt 2):399–407. doi: 10.1242/jcs.104.2.399. [DOI] [PubMed] [Google Scholar]
  15. Eichholtz T., Jalink K., Fahrenfort I., Moolenaar W. H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J. 1993 May 1;291(Pt 3):677–680. doi: 10.1042/bj2910677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Giuliano K. A., Kolega J., DeBiasio R. L., Taylor D. L. Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts. Mol Biol Cell. 1992 Sep;3(9):1037–1048. doi: 10.1091/mbc.3.9.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grand R. J., Grabham P. W., Gallimore M. J., Gallimore P. H. Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: independence from blood coagulation. EMBO J. 1989 Aug;8(8):2209–2215. doi: 10.1002/j.1460-2075.1989.tb08344.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hagag N., Halegoua S., Viola M. Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21. Nature. 1986 Feb 20;319(6055):680–682. doi: 10.1038/319680a0. [DOI] [PubMed] [Google Scholar]
  20. Hall A. Ras-related GTPases and the cytoskeleton. Mol Biol Cell. 1992 May;3(5):475–479. doi: 10.1091/mbc.3.5.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  22. Hamprecht B., Glaser T., Reiser G., Bayer E., Propst F. Culture and characteristics of hormone-responsive neuroblastoma X glioma hybrid cells. Methods Enzymol. 1985;109:316–341. doi: 10.1016/0076-6879(85)09096-6. [DOI] [PubMed] [Google Scholar]
  23. Harris A. K., Wild P., Stopak D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science. 1980 Apr 11;208(4440):177–179. doi: 10.1126/science.6987736. [DOI] [PubMed] [Google Scholar]
  24. Hawkins R. L., Seeds N. W. Effect of proteases and their inhibitors on neurite outgrowth from neonatal mouse sensory ganglia in culture. Brain Res. 1986 Nov 19;398(1):63–70. doi: 10.1016/0006-8993(86)91250-3. [DOI] [PubMed] [Google Scholar]
  25. Hirata K., Kikuchi A., Sasaki T., Kuroda S., Kaibuchi K., Matsuura Y., Seki H., Saida K., Takai Y. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem. 1992 May 5;267(13):8719–8722. [PubMed] [Google Scholar]
  26. Hordijk P. L., Verlaan I., van Corven E. J., Moolenaar W. H. Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway. J Biol Chem. 1994 Jan 7;269(1):645–651. [PubMed] [Google Scholar]
  27. Jalink K., Eichholtz T., Postma F. R., van Corven E. J., Moolenaar W. H. Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action. Cell Growth Differ. 1993 Apr;4(4):247–255. [PubMed] [Google Scholar]
  28. Jalink K., Moolenaar W. H. Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers. J Cell Biol. 1992 Jul;118(2):411–419. doi: 10.1083/jcb.118.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jalink K., van Corven E. J., Moolenaar W. H. Lysophosphatidic acid, but not phosphatidic acid, is a potent Ca2(+)-mobilizing stimulus for fibroblasts. Evidence for an extracellular site of action. J Biol Chem. 1990 Jul 25;265(21):12232–12239. [PubMed] [Google Scholar]
  30. Just I., Mohr C., Schallehn G., Menard L., Didsbury J. R., Vandekerckhove J., van Damme J., Aktories K. Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum. J Biol Chem. 1992 May 25;267(15):10274–10280. [PubMed] [Google Scholar]
  31. Kim S., Kikuchi A., Mizoguchi A., Takai Y. Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain. Brain Res Mol Brain Res. 1989 Nov;6(2-3):167–176. doi: 10.1016/0169-328x(89)90051-x. [DOI] [PubMed] [Google Scholar]
  32. Kitazawa T., Gaylinn B. D., Denney G. H., Somlyo A. P. G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem. 1991 Jan 25;266(3):1708–1715. [PubMed] [Google Scholar]
  33. Kruman I. I., Kostenko M. A., Gordon RYa, Popov V. I., Umansky S. R. Differentiation and apoptosis of murine neuroblastoma cells N1E115. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1309–1318. doi: 10.1006/bbrc.1993.1360. [DOI] [PubMed] [Google Scholar]
  34. Kumagai N., Morii N., Fujisawa K., Nemoto Y., Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J Biol Chem. 1993 Nov 25;268(33):24535–24538. [PubMed] [Google Scholar]
  35. Leevers S. J., Marshall C. J. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. doi: 10.1002/j.1460-2075.1992.tb05088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Li B. Q., Kaplan D., Kung H. F., Kamata T. Nerve growth factor stimulation of the Ras-guanine nucleotide exchange factor and GAP activities. Science. 1992 Jun 5;256(5062):1456–1459. doi: 10.1126/science.1604323. [DOI] [PubMed] [Google Scholar]
  37. Mizoguchi A., Kim S., Ueda T., Kikuchi A., Yorifuji H., Hirokawa N., Takai Y. Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain. J Biol Chem. 1990 Jul 15;265(20):11872–11879. [PubMed] [Google Scholar]
  38. Monard D. Cell-derived proteases and protease inhibitors as regulators of neurite outgrowth. Trends Neurosci. 1988 Dec;11(12):541–544. doi: 10.1016/0166-2236(88)90182-8. [DOI] [PubMed] [Google Scholar]
  39. Moodie S. A., Willumsen B. M., Weber M. J., Wolfman A. Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science. 1993 Jun 11;260(5114):1658–1661. doi: 10.1126/science.8503013. [DOI] [PubMed] [Google Scholar]
  40. Moolenaar W. H. LPA: a novel lipid mediator with diverse biological actions. Trends Cell Biol. 1994 Jun;4(6):213–219. doi: 10.1016/0962-8924(94)90144-9. [DOI] [PubMed] [Google Scholar]
  41. Morii N., Sekine A., Ohashi Y., Nakao K., Imura H., Fujiwara M., Narumiya S. Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein. J Biol Chem. 1988 Sep 5;263(25):12420–12426. [PubMed] [Google Scholar]
  42. Morii N., Teru-uchi T., Tominaga T., Kumagai N., Kozaki S., Ushikubi F., Narumiya S. A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J Biol Chem. 1992 Oct 15;267(29):20921–20926. [PubMed] [Google Scholar]
  43. Nakanishi S., Yamada K., Iwahashi K., Kuroda K., Kase H. KT5926, a potent and selective inhibitor of myosin light chain kinase. Mol Pharmacol. 1990 Apr;37(4):482–488. [PubMed] [Google Scholar]
  44. Narumiya S., Morii N. rho gene products, botulinum C3 exoenzyme and cell adhesion. Cell Signal. 1993 Jan;5(1):9–19. doi: 10.1016/0898-6568(93)90003-5. [DOI] [PubMed] [Google Scholar]
  45. Narumiya S., Sekine A., Fujiwara M. Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product. J Biol Chem. 1988 Nov 25;263(33):17255–17257. [PubMed] [Google Scholar]
  46. Nemoto Y., Namba T., Kozaki S., Narumiya S. Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli. J Biol Chem. 1991 Oct 15;266(29):19312–19319. [PubMed] [Google Scholar]
  47. Nishiki T., Narumiya S., Morii N., Yamamoto M., Fujiwara M., Kamata Y., Sakaguchi G., Kozaki S. ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells. Biochem Biophys Res Commun. 1990 Feb 28;167(1):265–272. doi: 10.1016/0006-291x(90)91760-p. [DOI] [PubMed] [Google Scholar]
  48. Noda M., Ko M., Ogura A., Liu D. G., Amano T., Takano T., Ikawa Y. Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line. Nature. 1985 Nov 7;318(6041):73–75. doi: 10.1038/318073a0. [DOI] [PubMed] [Google Scholar]
  49. Paterson H. F., Self A. J., Garrett M. D., Just I., Aktories K., Hall A. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990 Sep;111(3):1001–1007. doi: 10.1083/jcb.111.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Qiu M. S., Green S. H. NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation. Neuron. 1991 Dec;7(6):937–946. doi: 10.1016/0896-6273(91)90339-2. [DOI] [PubMed] [Google Scholar]
  51. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  52. Seeds N. W., Gilman A. G., Amano T., Nirenberg M. W. Regulation of axon formation by clonal lines of a neural tumor. Proc Natl Acad Sci U S A. 1970 May;66(1):160–167. doi: 10.1073/pnas.66.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sekine A., Fujiwara M., Narumiya S. Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. J Biol Chem. 1989 May 25;264(15):8602–8605. [PubMed] [Google Scholar]
  54. Sellers J. R. Regulation of cytoplasmic and smooth muscle myosin. Curr Opin Cell Biol. 1991 Feb;3(1):98–104. doi: 10.1016/0955-0674(91)90171-t. [DOI] [PubMed] [Google Scholar]
  55. Settleman J., Albright C. F., Foster L. C., Weinberg R. A. Association between GTPase activators for Rho and Ras families. Nature. 1992 Sep 10;359(6391):153–154. doi: 10.1038/359153a0. [DOI] [PubMed] [Google Scholar]
  56. Shirataki H., Kaibuchi K., Sakoda T., Kishida S., Yamaguchi T., Wada K., Miyazaki M., Takai Y. Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol Cell Biol. 1993 Apr;13(4):2061–2068. doi: 10.1128/mcb.13.4.2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Skaper S. D., Selak I., Varon S. Serum- and substratum-dependent modulation of neuritic growth. J Neurosci Res. 1983;9(4):359–369. doi: 10.1002/jnr.490090402. [DOI] [PubMed] [Google Scholar]
  58. Smalheiser N. R. Acute neurite retraction elicited by diverse agents is prevented by genistein, a tyrosine kinase inhibitor. J Neurochem. 1993 Jul;61(1):340–343. doi: 10.1111/j.1471-4159.1993.tb03573.x. [DOI] [PubMed] [Google Scholar]
  59. Suidan H. S., Stone S. R., Hemmings B. A., Monard D. Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors. Neuron. 1992 Feb;8(2):363–375. doi: 10.1016/0896-6273(92)90302-t. [DOI] [PubMed] [Google Scholar]
  60. Thomas S. M., DeMarco M., D'Arcangelo G., Halegoua S., Brugge J. S. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell. 1992 Mar 20;68(6):1031–1040. doi: 10.1016/0092-8674(92)90075-n. [DOI] [PubMed] [Google Scholar]
  61. Tigyi G., Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochromocytoma cells. J Biol Chem. 1992 Oct 25;267(30):21360–21367. [PubMed] [Google Scholar]
  62. Tominaga T., Sugie K., Hirata M., Morii N., Fukata J., Uchida A., Imura H., Narumiya S. Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho. J Cell Biol. 1993 Mar;120(6):1529–1537. doi: 10.1083/jcb.120.6.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
  64. Warne P. H., Viciana P. R., Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul 22;364(6435):352–355. doi: 10.1038/364352a0. [DOI] [PubMed] [Google Scholar]
  65. Windebank A. J., Blexrud M. D. Characteristics of neurite outgrowth from rat spinal ganglia: effects of serum and segmental level. J Neuropathol Exp Neurol. 1986 Nov;45(6):683–691. doi: 10.1097/00005072-198611000-00006. [DOI] [PubMed] [Google Scholar]
  66. Wood K. W., Sarnecki C., Roberts T. M., Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]
  67. Ziller C., Le Douarin N. M. Neuronal differentiation in cultured neural crest cells: the effect of serum on neurite outgrowth. Birth Defects Orig Artic Ser. 1983;19(4):251–261. [PubMed] [Google Scholar]
  68. van Corven E. J., Hordijk P. L., Medema R. H., Bos J. L., Moolenaar W. H. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1257–1261. doi: 10.1073/pnas.90.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. van der Bend R. L., Brunner J., Jalink K., van Corven E. J., Moolenaar W. H., van Blitterswijk W. J. Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid. EMBO J. 1992 Jul;11(7):2495–2501. doi: 10.1002/j.1460-2075.1992.tb05314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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